CN112540179B

CN112540179B - ELISA kit for testing content of ApoE4 protein

Info

Publication number: CN112540179B
Application number: CN202010785800.4A
Authority: CN
Inventors: 王小川; 王喆; 曾宽; 吴刚
Original assignee: Wuhan Tiande Biotechnology Co ltd
Current assignee: Wuhan Tiande Biotechnology Co ltd
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2023-07-07
Anticipated expiration: 2040-08-06
Also published as: CN112540179A

Abstract

The invention provides an ELISA kit for testing the content of ApoE4 protein, which comprises the following components: solid phase carrier containing capture antibody, sample diluent, standard protein the enzyme-labeled primary antibody pan ApoE, a chromogenic substrate and a stop solution; wherein the capture antibody is an anti-APOE 4 point mutation monoclonal antibody. The invention has the beneficial effects that: the double-sandwich monoclonal antibody ELISA kit provided by the invention can specifically identify the APOE4 point mutant protein, and can be used for screening by two screening modes of APOE4-Arg, APOE4-Cys epitope peptide and APOE4-Arg and APOE4-Cys protein produced by coated genetically engineered cells, wherein the two screening modes are synthesized by a coated solid phase method, and the double-sandwich monoclonal antibody ELISA kit has good sensitivity, specificity and accuracy.

Description

ELISA kit for testing content of ApoE4 protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an ELISA kit for testing the content of ApoE4 protein.

Background

Alzheimer's Disease (AD), also known as Alzheimer's disease, is an age-related neurodegenerative disease that is manifested clinically by memory dysfunction and alterations in behavior. As a progressive disease, the most important physiological features of AD are the large brain regions experiencing progressive dysfunction, neuronal loss and loss of synapses. The study by batman et al demonstrated that the brains of patients began to change gradually 10 to 20 years prior to the onset of AD. These changes in functionality are often difficult to reverse, which also results in current dilemma of AD treatment. Thus, detection of early AD is probably the most effective means of preventing and treating AD.

Detection of blood biomarkers is the preferred mode of detection for a number of diseases due to its inexpensive, easy to handle, and non-invasive advantages. Whereas in AD numerous proteins have been shown to have significant changes in the plasma of AD patients, such as BDNF, AGT, IGFBP-2, OPN, etc., suggesting that plasma protein content detection may be a valuable way for early diagnosis of AD.

And apolipoprotein E (ApoE) has been shown to be associated with the development of AD. The apolipoprotein (Apo) E gene is divided into three allelic genotypes epsilon 2, epsilon 3, epsilon 4, respectively encoding three isomers of apoE2, apoE3 and apoE4. The only difference between the three different subtypes of ApoE is the amino acids at positions 112 and 158 of their primary structure, namely ApoE2 (112 cys,158 cys), apoE3 (112 cys,158 arg), apoE4 (112 arg,158 arg). Among them, high expression of the ApoE.epsilon.4 gene has been recognized as one of the biggest risk factors for AD. Immunohistochemical analysis of Sporadic AD (SAD) and late Familial AD (FAD), which account for the majority of the disease in Duke university 1990, found the presence of ApoE4 in senile plaques and neurofibrillary tangles. Studies have shown that both sporadic and familial late-onset AD are associated with ε 4. The probability of developing late onset AD is 3 times higher for heterozygote epsilon 4 carriers than for non-carriers, and 10-20 times higher for homozygous epsilon 4 carriers than for non-carriers. The epsilon 4 gene was found to correlate with patient age and age of onset by carwfodr et al. Whereas in early diagnosis, if an individual carries ApoE4, the diagnosis of AD is increased by at least 5% to 10% confidence, especially for differential diagnosis of different dementia types. Meanwhile, if the patient has AD related behavioral phenotype and carries ApoE4, the accuracy of diagnosing AD can reach 94% -98%. These all suggest: detection of ApoE4 genotype is probably one of the most valuable indicators for early AD diagnosis.

However, at present, the detection of the ApoE genotype at home and abroad mainly passes through a second-generation high-throughput sequencing system, so that the cost is high and the accuracy is difficult to ensure. The report results of the national tumor second generation sequencing laboratory quality assessment at the end of 2015 show that 55% of the laboratory sequencing results are disqualified and 22% of the laboratory quality assessment is 0 score. These lead to ApoE4 genotype detection which is difficult to popularize in AD diagnosis.

And a change in genotype will result in a change in protein expression. It is reported in literature that differences in ApoE alleles can cause changes in serum ApoE-inhibiting concentrations in AD patients and cause abnormal blood lipid metabolism in AD patients; the serum apoE4 concentration of AD patients is obviously increased compared with normal people. It is suggested that the detection of ApoE4 protein content in serum may be of great importance in the diagnosis of early AD. At present, no ApoE4 protein detection kit is marketed at home and abroad. Meanwhile, the protein kit detection has the advantages of high accuracy and low cost. Therefore, researchers consider that the research and development of the serum ApoE4 protein detection kit can provide powerful evidence for early AD diagnosis, and has certain practical significance and economic benefit.

The application of monoclonal antibody technology in disease diagnosis and drug development is a research hotspot in the current medical field, and provides a new way for the treatment of various diseases.

At present, the ApoE4 monoclonal antibody used for detection cannot completely distinguish between ApoE2 and ApoE3 types, so that the specificity of the ApoE4 monoclonal antibody for ApoE4 detection is not strong, and further, an ApoE4 reagent detection product with strong specificity cannot be developed.

Disclosure of Invention

In order to develop a rapid, sensitive and high-specificity APOE4 detection kit, the invention provides an ELISA kit for testing the content of ApoE4 protein.

The specific technical scheme is as follows:

an ELISA kit for testing ApoE4 protein content, which is different in that the ELISA kit for testing ApoE4 comprises:

the method comprises the steps of coating a solid phase carrier containing a capture antibody, a sample diluent, a standard substance protein, an enzyme-labeled primary antibody panApoE, a chromogenic substrate and a stop solution;

wherein the capture antibody is an anti-APOE 4 point mutation monoclonal antibody, and the heavy chain amino acid sequence of the anti-APOE 4 point mutation monoclonal antibody is as follows: 3, the light chain amino acid sequence of the anti-APOE 4 point mutant monoclonal antibody is as shown in SEQ NO: 4.

Further, the anti-APOE 4 point mutation monoclonal antibody is prepared by a hybridoma cell strain, wherein the hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is as follows: CCTCCNO: C2020121.

further, the standard protein is ApoE4 standard freeze-dried powder.

Further, the sample diluent is 0.01mol/L phosphate buffer solution and contains 1% of BSA, 0.05% of Tween-20 and 1mg/L gentamicin by mass percent.

Further, the termination liquid is H of 2mol/L ₂ SO ₄ 。

Further, the enzyme-labeled primary antibody is horseradish peroxidase-labeled murine monoclonal antibody panApoE.

Further, the color developing agent is TMB substrate color developing agent.

Compared with the prior art, the invention has the beneficial effects that: the double-sandwich monoclonal antibody ELISA kit provided by the invention can specifically identify the APOE4 point mutant protein, and can be used for screening by two screening modes, namely APOE4-Arg, APOE4-Cys protein and APOE4-Arg and APOE4-Cys protein produced by coated genetically engineered cells, wherein the APOE4-Arg, the APOE4-Cys protein and the APOE4-Cys protein are synthesized by a coated solid phase method, and the kit has good sensitivity, specificity and accuracy.

Drawings

FIG. 1 is an electropherogram of purified antibody SDS-PAEG, wherein MW: represents molecular weight; lane 1 shows ACE8 denatured by not boiling; lane 2; indicating that ACE8 is denatured by boiling;

FIG. 2 shows Western blot analysis of extracts of transfected HeLa cells expressing full-length human ApoE2 (hApoE 2), apoE3 (hApoE 3) or ApoE4 (hApoE 4), wherein the primary antibody used in the upper panel is the monoclonal antibody produced by hybridoma ACE8 of the present invention, and the primary antibody used in the lower panel is the panapoE antibody;

FIG. 3 is a graph showing the results of an immunofluorescence assay of primary rat hippocampal nerve cells using ApoE4 (ACE 8) mouse mAb mAb murine mAb;

FIG. 4 is a photograph of human brain glioma U87 cell immunofluorescent cell staining with ApoE4 (ACE 8) mouse mAb mAb;

FIG. 5 is a graph of the results of immunohistochemical analysis of paraffin-embedded human skin using ApoE4 (ACE 8) mouse mAb, wherein A is control peptide and B is antigen-specific peptide;

FIG. 6 is a graph showing the results of immunohistochemical analysis of paraffin-embedded human kidney tissue sections using ApoE4 (ACE 8) mouse mAb murine mAb;

FIG. 7 is a graph showing the results of immunohistochemical analysis of paraffin-embedded human spleen tissue sections using ApoE4 (ACE 8) mouse mAb murine mAb;

fig. 8 is a graph of results of immunohistochemical analysis of paraffin-embedded human colon tissue sections using ApoE4 (ACE 8) mouse mAb murine mAb.

Detailed Description

The principles and features of the present invention are described below in connection with the following examples, which are set forth to illustrate, but are not to be construed as limiting the scope of the invention.

EXAMPLE 1 immunocomplex preparation

(1) The 16 peptide of APOE4 protein (amino acid sequence 109-124 of APOE4 protein: EDVRGRLVQYRGEVQA, shown in SEQ ID NO. 5) was synthesized by solid phase method on an ABI polypeptide synthesizer (431A) in the United states, using Fmoc (9-fluorenylmethoxycarbonyl) protocol, and the synthesis procedure was performed according to the ABI polypeptide synthesis manual. Purifying by high performance liquid chromatography, and performing sequence identification to obtain the epitope peptide of the APOE4 protein by mass spectrometry.

(2) And coupling the epitope peptide of the prepared ApoE4 protein with KLH through a coupling reagent Sulfo-SMCC to obtain the immune complex.

The above can be achieved by using reagents, instruments, etc. common in the art.

EXAMPLE 2 monoclonal antibody preparation

(1) Immunization of animals

Six female Balb/c inbred mice of 6-8 weeks of age were used for five immunization injections according to the following immunization protocol.

Primary immunization: on day 0, 0.2ml of emulsion was subcutaneously injected into the groin of each mouse using a 2ml syringe, wherein the emulsion was an immunocomplex solution and Freund's complete adjuvant at 1:1, wherein the emulsion contains 30-50 mug immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

Secondary immunization: on day 21, 0.2ml of an emulsion was subcutaneously injected into the groin of each mouse using a 2ml syringe, wherein the emulsion was an immunocomplex solution and Freund's incomplete adjuvant at a rate of 1:1, wherein the emulsion contains 30-50 mug immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

Third immunization: on day 42, 0.2ml of emulsion was subcutaneously injected into the groin of each mouse using a 2ml syringe, wherein the emulsion was an immunocomplex solution and Freund's incomplete adjuvant at 1:1, wherein the emulsion contains 30-50 mug immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

Fourth immunization: on day 63, 0.2ml of emulsion was subcutaneously injected into the groin of each mouse using a 2ml syringe, wherein the emulsion was an immunocomplex solution and Freund's incomplete adjuvant at a rate of 1:1, wherein the emulsion contains 30-50 mug immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

Fifth immunization: on day 78, each mouse was injected intravenously with 0.2ml of an emulsion using a 2ml syringe, wherein the emulsion was an immunocomplex solution and Freund's incomplete adjuvant at 1:1, wherein the emulsion contains 30-50 mug immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

Immune serum titers were tested by indirect ELISA on day 1, day 52, and day 73, respectively, where the titer on day 73 reached 128K or higher, the mouse tail vein was subjected to impact immunization on day 78, and after 3 days (day 81) mouse spleen B cells were taken for fusion.

(2) Preparation of hybridoma cells

Preparation of mouse myeloma cell SP 2/0: SP2/0 myeloma cell lines from BALB/C mice were passaged in 10% FBS-DMEM medium, and cultured inContaining 5% CO ₂ Culturing in an incubator at 37 ℃ with saturated humidity. Passaging is performed one day before fusion to ensure that cells enter the logarithmic phase of growth when fused.

Preparation of feeder cells: macrophages from the abdominal cavity of the mice were used as feeder cells. Normal BALB/c mice were collected, the eyeballs were removed to collect blood, and serum was isolated as a negative control serum for antibody detection. Meanwhile, killing the mice through cervical dislocation, soaking in 75% alcohol for 5 minutes, fixing on a dissecting table plate, lifting the skin at the left side abdomen, viewing spleen, changing the forceps of the eye scissors, shearing peritoneum in an ultra clean bench by using a sterile surgical scissors, taking out the spleen, placing in a plate, and carefully peeling off surrounding connective tissues. The spleen was pressed with a plunger to disperse the cells, and the cells were purged several times with 10ml of incomplete medium to prepare a single cell suspension. Transferring into 50ml centrifuge tube, centrifuging at 1000r/min for 5-10 min, centrifuging with incomplete culture medium for 1-2 times, suspending cells in 10ml incomplete culture medium, mixing, collecting the suspension, adding blue dye solution of phenol, and counting. Typically 1X 10 per mouse is available ⁸ -2.5×10 ⁸ Spleen cells were individually prepared by adding the above cell suspension to a 96-well plate in an amount of 0.1ml (equivalent to 2 drops) per well, and then placing the mixture at 37℃in 4% CO ₂ Is cultured in an incubator of (a).

Preparation of immune spleen cells: balb/c mice immunized were necked or CO ₂ Killing, soaking in 75% ethanol solution for 5min, aseptically opening abdomen in a super clean bench, taking out spleen, and cleaning once with 5ml of incomplete culture solution of RPM 1640; placing a stainless steel mesh in a plate containing 20ml of incomplete culture solution, transferring the spleen onto a screen, lightly grinding the spleen with an inner core of a syringe, sucking culture medium liquid in the plate, lightly washing the stainless steel screen, and allowing spleen cells to completely enter the solution through meshes; transferring the spleen cell solution into a50 ml centrifuge tube, adding 15-20ml of incomplete culture solution, and mixing; centrifuging for 10min at 1200r/min, and discarding supernatant; the cell pellet is centrifuged and washed once by using an incomplete culture solution, and then resuspended, and trypan blue staining is used for counting living cells. The normal immune spleen cell volume is about 2 times that of normal spleen, and each mouse can obtain 1.0X10 ⁸ -2.5×10 ⁸ Spleen cells.

Cell fusion: taking myeloma cells SP2/0 in logarithmic growth phase, centrifuging at 1000r/min for 5 minutes, discarding supernatant, re-suspending cell sediment by using an incomplete culture solution, uniformly mixing, dyeing by using trypan blue or counting cells, taking required cells, and washing for 2 times by using the incomplete culture solution; washing the prepared immune spleen cells with an incomplete culture solution for 2 times; adding myeloma cells and spleen cells into the same 50ml centrifuge tube according to the ratio of 1:10 or 1:5, adding incomplete culture solution to 30-40ml, and fully and uniformly mixing; centrifuging at 1200r/min for 10min, discarding supernatant, and lightly flicking the bottom of the centrifuge tube to loosen the cell pellet into uniform paste. Fusion at room temperature: the centrifuge tube was uniformly turned on one hand, 45% PEG60001ml preheated at 37℃was pipetted on the other hand with a 1ml pipette and added uniformly and slowly along the wall of the rotating centrifuge tube for a period of about 60 seconds, then the whole cell suspension was immediately gently aspirated into the pipette for a period of 30 seconds, left to stand for 30 seconds, then gently blown into the centrifuge tube for a period of about 30 seconds, and the incomplete culture broth preheated to 37℃was immediately added in five minutes at volumes of 1, 2, 4, 8 and 10ml per minute (25 ml total) and was quenched by dilution of PEG. Centrifugation at 800r/min for 6 min, discarding supernatant, adding appropriate amount of 20% FCS-RPMI1640 complete culture solution, gently suspending the fused cells, and no forceful blowing of cells is needed. The complete broth amount was calculated as 10ml of a 96 well cell culture plate.

The fused cell suspension was added to a 96-well cell culture plate with feeder cells spread therein at 100. Mu.l/well, 37℃and 5% CO ₂ Culturing in an incubator. Typically, 4-6 96 well plates can be added after 1 immunized mouse spleen cells are fused.

(3) Selection and cloning of hybridoma cells

1/2 of the culture medium (HAT) was changed every 4 days, and after 10 days, HT-containing selective medium was used instead. The fused hybridoma cells were cultured in a selective medium containing HT for about two weeks. When the cell colonies grow to a proper size (the size of the cell clone is preferably that of the cell clone which occupies one field of view when observed under a 10-time objective lens), the culture supernatant is sucked, and after proper dilution, indirect ELISA is performed to screen positive clones.

a. Preliminary screening

The epitope polypeptide APOE4-112Arg (amino acid sequence of 109-124 of APOE4 protein: EDVRGRLVQYRGEVQA is used as positive screening) and APOE4-112Cys (EDVCGRLVQYRGEVQA is used as negative screening) are coated, the epitope polypeptide used as negative screening is synthesized by adopting a solid phase method, the solid phase method is adopted for screening, the indirect ELISA method is adopted for positive reaction on the APOE4-112Arg polypeptide, the cell strain which does not react on the APOE4-112Cys is the positive cell strain of the primary screening, and the positive cell strain can be further screened according to the step b.

b. Secondary screening

The nucleotide sequences containing the APOE4-112 Cys-encoded protein, i.e., APOE2 (112 Cys,158 Cys) protein, APOE3 (112 Cys,158 Arg) protein and APOE4-112 Arg-encoded protein, i.e., APOE4 (112 Arg,158 Arg) protein, were cloned (wherein the nucleotide sequence encoding the APOE2 protein is shown as SEQ ID No. 6, the amino acid sequence encoding the APOE2 protein is shown as SEQ ID No. 9, the nucleotide sequence encoding the APOE3 protein is shown as SEQ ID No. 7, the amino acid sequence encoding the APOE2 protein is shown as SEQ ID No. 10, the amino acid sequence encoding the APOE4 protein is shown as SEQ ID No. 8), the plasmid encoding the APOE2 protein is shown as SEQ ID No. 11) onto a pcdna3.1 (+) full length expression plasmid and the plasmid was prepared according to standard molecular biological methods, HEK293 cells (purchased from ATCC, usa) were infected by transient transfection methods, and the cells were lysed, and the expressed proteins were purified.

The three proteins are coated, and the primary screened positive cell strain is further screened by adopting an indirect ELISA method, has positive reaction on the APOE4-112Arg protein, and the cell strain which does not react on the APOE4-112Cys protein is regarded as a positive cell strain.

The positive cell lines selected were cloned by limiting dilution. Clones of the single colonies were selected and positive cell lines were further determined using the methods of a and b.

Preparing hybridoma cell suspension: gently blowing off the hybridoma cells in the ELISA detection positive holes, sucking the hybridoma cells into a 10mL centrifuge tube by using a pipette, counting, calculating, diluting the cell concentration to 200/mL by using a complete culture medium, taking out 2mL, adding the 2mL into 2mL of complete culture medium containing feeder cells, and changing the concentration of the hybridoma cells to 100/mL;

limiting dilution: the hybridoma cell suspension was added at 100. Mu.L per well to two

rows

1 and 2 of a 96-well cell culture plate, and the concentration of the hybridoma cells in these two rows was 10 per well. Adding equal volume of feeder cell suspension into the rest cell suspension, mixing, adding into the 3 rd and 4 th rows, wherein the concentration of hybridoma cells in the two rows is 5 per hole, repeating the steps, and the concentration of hybridoma cells in the 5 th and 6 th rows is 2.5 per hole, and the concentration of hybridoma cells in the 7 th and 8 th rows is 1.25 per hole. After 3 days of culture, the growth of the cells was observed under a microscope, and 100. Mu.L of the culture medium on the culture plate was carefully aspirated, and 100. Mu.L of the complete medium was further added. The Kong Zhongshang clear is detected by indirect ELISA, and the cell culture well with high titer is selected for re-cloning, and a monoclonal well is selected as much as possible. Until two subsequent cloned monoclonal wells were all positive.

Cell fusion 6 hybridoma cell lines capable of stably secreting anti-APOE 4 point mutant monoclonal antibodies were obtained, see Table 1 below. The antibody secreted by the hybridoma cell strain ACE8 has optimal effect when being used for detecting the APOE4 protein, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2020121 at the month of 7 and the day of 22 in 2020.

Table 1 hybridoma cell strain capable of stably secreting anti-ApoE 4 protein monoclonal antibody obtained by screening

Hybridoma cell strain	Hybridoma cell strain	Hybridoma cell strain
			ACE8	ACE4	ACE14
ACE23	ACE36	ACE72

The hybridoma cell line supernatant was assayed by indirect ELISA, and 2-fold stepwise dilution was performed from the 3 rd well to the 10 th well with 0.01 mM PBS buffer of pH 7.2. The 1 st hole takes the serum of the mice diluted to 100 times as positive control when in fusion, the 2 nd hole takes RPMI1640 complete culture solution as negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the positive control is effective as a detection system, and when the OD value is more than or equal to 2 times of the OD value of the negative control, the positive control is positive, otherwise the negative control is negative. The dilution ratio corresponding to the lowest positive hole of the detection value is the culture supernatant titer of the hybridoma cell strain, and the culture supernatant titer of the hybridoma cell strain can reach 4096K (K, 1000,4096K, 4096000) as shown in the table 2 below.

TABLE 2 results of hybridoma cell line supernatant titers

(4) Ascites production and purification of monoclonal antibodies

Induction of ascites: before inoculating hybridoma cells, 1 week before inoculating the hybridoma cells, 0.3-0.5 ml/liquid paraffin is used for inoculating into the abdominal cavity of the mice, so that the mice are sensitized. Hybridoma cells diluted with PBS or serum-free medium 7-10 days later were inoculated into the peritoneal cavity of mice, each 3X 10 ⁵ 0.5ml. After inoculation, the generation condition of the ascites of the mice is observed every day, if the abdomen is obviously enlarged, when the mice are touched by hands, the skin is stressed, the mice can be killed by pulling the neck, the ascites is sucked into a 15ml centrifuge tube by a dropper, and 1-5ml of ascites can be obtained by one mouse. Centrifuging the collected ascites to obtain supernatant, taking a small sample, and storing in a refrigerator below-20deg.C for use.

Purification of monoclonal antibodies: and purifying the collected ascites by an ammonium sulfate precipitation method. SDS-PAGE performed purity validation and quantification of the antibodies, and the results are shown in FIG. 1.

The specific purification steps are as follows: 1) Adding 2 parts of PBS into 1 part of ascites for dilution; 2) Then adding 0.277g ammonium sulfate/ml into the ascites diluent according to the total volume after dilution (the process of adding ammonium sulfate needs to be carried out in an ice-water bath, and adding is carried out in multiple times while shaking, and the adding is completed in about 30 minutes); 3) Then standing at 4 ℃ for two hours or overnight; 4) Centrifuging at 12000rpm,10min or 3000rpm,20min, discarding supernatant, and redissolving with PBS to original volume; 5) Repeating the steps 2 and 3; 6) Centrifuging at 12000rpm,10min or 3000rpm,20min, discarding supernatant, dissolving with PBS, measuring concentration, detecting purity by SDS-PAGE, and adding 40-50% glycerol after detection.

(5) Determination of ascites titer

Ascites titers were determined by indirect ELISA from wells 3 to 10 by 2-fold stepwise dilution with 0.01 mM PBS buffer pH 7.2. The 1 st hole takes the serum of the mice diluted to 100 times as positive control when in fusion, the 2 nd hole takes RPMI1640 complete culture solution as negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the positive control is effective as a detection system, and when the OD value is more than or equal to 2 times of the OD value of the negative control, the positive control is positive, otherwise the negative control is negative. The dilution ratio corresponding to the positive well with the lowest detection value is the ascites titer which can reach 4096K (K, 1000,4096K, 4096000).

(6) Antibody titer determination

The purified antibody was uniformly diluted to 1mg/ml with 0.01 mM PBS buffer at pH7.2, and then diluted 100-fold as the initial 1 st well, followed by 2-fold stepwise dilution from 2 nd well to 10 th well. The 11 th hole takes the serum of the mice diluted to 100 times as positive control when in fusion, the 12 th hole takes RPMI1640 complete culture solution as negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, and the positive control is the effective detection system, the antibody titer judgment standard: the curve is plotted on the abscissa of log (dilution) and on the ordinate of OD values, the equation of the curve is y=min+ (max-min)/(1+10 ++1 (log ec 50-x) ×hillslope)), the curve is fitted with sigmaplot software, and the titer=10 log ec50 is taken. The results showed that the anti-APOE 4 point mutant monoclonal antibody titer was 4096K (K, 1000,4096K 4096000).

(7) Subtype identification of monoclonal antibodies by analysis with a mouse monoclonal antibody immunoglobulin typing kit from Serotec company. The purified monoclonal antibody is properly diluted and then detected, and the operation is strictly carried out according to the instruction of the kit. The test results are shown in Table 3 below (data source A: absorbance 450 nm):

TABLE 3 subtype detection results

The monoclonal antibody secreted by the hybridoma cell strain ACE8 is gamma 1 type, and has high specificity and sensitivity with the antigen peptide (109-124 amino acid sequences) of APOE4. Hybridoma cell line ACE8 has been preserved by China Center for Type Culture Collection (CCTCC); address: university of martial arts in chinese; preservation date: 7 months 22 days 2020; the preservation number is: CCTCCNO: C2020121.

EXAMPLE 3 identification of monoclonal antibodies

(1) Immunoblot identification

Respectively constructing pcDNA3.1 (+) full-length expression plasmids containing nucleotide sequences of APOE4-112Cys, namely ApoE2 (112 Cys,158 Cys) protein, apoE3 (112 Cys,158 Arg) protein and APOE4-112Arg, namely ApoE4 (112 Arg,158 Arg) protein, respectively transfecting HEK-293 cells with the plasmids, carrying out WesternBlotting detection on culture cell lysates by using the monoclonal antibodies, and presenting target bands at corresponding positions, thereby indicating that the expression of the ApoE4-112Arg protein is detected. The method comprises the following specific steps:

a. SDS polyacrylamide gel electrophoresis: methods are described in F.Osbert et al, guidelines for the experiments in fine-programming molecular biology (scientific Press 1998). The electrophoresis was terminated by 10% separation gel and 5% concentration gel at a voltage of 150V with bromophenol blue dye band 1.5 cm from the bottom edge of the gel.

b. Electrotransfer: methods are described in F.Osbert et al, guidelines for the experiments in fine-programming molecular biology (scientific Press 1998). It was transferred to PVDF (0.45 μm) membrane by electrotransfer.

c. Immunoblotting: after the electrotransfer membrane is finished, the membrane is blocked for 1 hour in 5% nonfat milk powder blocking solution at room temperature, the monoclonal antibody (1 mug/ml) prepared by the invention is used as a primary antibody, the membrane is incubated for 2 hours at room temperature or is reacted overnight at 4 ℃, and the membrane is washed 3 times by TBST (TBS added with 0.5% Tween-20) for 10 minutes each time. The goat anti-mouse IgG marked by horseradish peroxidase is used as a secondary antibody, reacted for 2 hours at room temperature, washed by the method and acted by ECL for 1min, and then is exposed and imaged by a full-automatic chemiluminescence imager (Bio-Rad Versadoc5000 MP).

The results are shown in FIG. 2, FIG. 2 shows Western blot analysis of transfected HeLa cell extracts expressing full-length human ApoE2 (hApoE 2), apoE3 (hApoE 3) or ApoE4 (hApoE 4), wherein the primary antibody used in the upper panel is the monoclonal antibody produced by hybridoma cell ACE8 of the present invention and the primary antibody used in the lower panel is the panapoE antibody (available from Eimer biosciences, inc.). ApoE2 (112 Cys,158 Cys), apoE3 (112 Cys,158 Arg) were used as controls, and neither was bound to the antibody, but ApoE4 (112 Arg,158 Arg) was specifically bound to the antibody.

(2) Immunofluorescent cell staining identification

Respectively constructing pcDNA3.1 (+) full-length expression plasmids containing nucleotide sequences of APOE4-112Cys, namely ApoE2 (112 Cys,158 Cys) protein, apoE3 (112 Cys,158 Arg) protein and APOE4-112Arg, namely ApoE4 (112 Arg,158 Arg) protein, respectively transfecting primary rat hippocampal nerve cells/human brain glioma U87 cells with the plasmids, and verifying an antibody positive to the APOE4-112Arg according to an immunofluorescence technical flow, wherein the results are shown in figures 3 and 4.

(3) Immunohistochemical identification

Sections of human skin, human kidney, human spleen, colon (all obtained from the university of martial arts, south-middle school hospital) of a part of patients suffering from senile dementia were taken, and the sections were treated as follows and observed under a microscope:

tissue of fixing: the sections were baked in a 60 ℃ incubator for 60 minutes prior to dewaxing.

Dewaxing: the sections were soaked with xylene for 10 minutes, replaced with xylene and soaked for another 10 minutes.

Hydration: soaking in absolute ethanol for 5 minutes respectively; soaking in 95% ethanol for 5min; soaking in 85% ethanol for 5min; soaking in 75% ethanol for 5 min.

Washing: ddH2O was soaked for 5 minutes and washed 3 times.

Antigen retrieval (boiling method): adding 10mmol/ml citrate buffer (pH 6.0) to the pressure cooker, heating to boil, placing the slices on a heat-resistant plastic slice frame, placing into the cooker, covering the cooker cover, fastening a pressure valve, continuing heating, starting timing when the pressure valve is sprung up for air injection, stopping heating after 1.5 min, opening the cooker cover after the pressure is zeroed, and taking out the slices after the solution is cooled to room temperature.

Washing: ddH ₂ O was soaked for 5 minutes, washed 2 times, and PBST (500 mL10 XPBS+ 4500mLddH2O+5mL Tween-20) was soaked for 5 minutes, washed 2 times.

Inactivating the enzyme: mu.l of 3% H2O2-PBS was added dropwise to each plate, and the plate was treated at room temperature for 15 minutes.

Washing: PBST was soaked for 5 minutes and washed 3 times.

Adding an antibody: 100 μl of diluted primary antibody (anti-APOE 4 point mutation monoclonal antibody, i.e., ACE 8-produced monoclonal antibody) was added and incubated in a wet box at 37deg.C for 1 hour.

Washing: PBST was soaked for 5 minutes and washed 3 times.

Postblocking: add one drop of 50 μl of the histochemical kit (Maixin ready-to-use histochemical kit) reagent A and incubate in a wet box for 30 minutes at room temperature.

Washing: PBST was soaked for 5 minutes and washed 3 times.

Adding enzyme-labeled secondary antibodies: one drop of 50 μl of the histochemical kit reagent B (HRP-labeled secondary antibody) was added, and incubated at 37℃for 30 minutes.

Washing: PBST was soaked for 5 minutes and washed 3 times.

Color development: DAB method: adding 0.85ml distilled water into 50 μl DAB color development kit (purchased from Fuzhou Michaelsholtzia Biotechnology Co., ltd.) reagent A, and mixing; adding 50 μl of DAB chromogenic kit reagent B, and mixing; adding 50 μl of DAB chromogenic kit reagent C, and mixing; and (5) light shielding. Drop onto sections, 100 μl of each wet box, and develop color for 5 minutes.

Terminating the color development: the color reaction was stopped with distilled water.

Counterstaining: the sections were placed in hematoxylin dye, stained for 30min, and rinsed with distilled water.

Sealing piece: DAB method: put into hydrochloric acid methanol solution and immediately washed clean with distilled water. Soaking in 75% ethanol for 5min; soaking in 85% ethanol for 5min; soaking in 95% ethanol for 5min; soaking in absolute ethanol for 5 minutes. Soaking with xylene for 10min, replacing xylene, and soaking for 10min. After air drying, add neutral gum to the sections and cover the slides.

And (3) observation: the results are shown in fig. 5, 6, 7 and 8, wherein in fig. 5, A is a control peptide, the amino acid sequence of which is VERDVGRLRVQQGYEA, B is an antigen-specific peptide, the amino acid sequence of which is EDVRGRLVQYRGEVQA, and both the control peptide and the antigen-specific peptide are synthesized by a solid phase method.

(4) Hybridoma cell strain ACE8 and anti-APOE 4 point mutation monoclonal antibody sequence determination

Extracting total RNA of hybridoma cell strain ACE8, reversely transcribing into cDNA by using a general primer, amplifying light chain and heavy chain of the antibody, separating the light chain and the heavy chain, cloning the light chain and the heavy chain to a standard cloning vector for expression, identifying the light chain and the heavy chain by single colony PCR, selecting 5 single colonies with correct length of the light chain and the heavy chain for sequencing, and considering the real sequence of the antibody if the sequencing results are almost the same after 5 times. (sequencing Process and antibody Mass expression commission done by Nanjing Jinsri Biotech Co., ltd.)

Gene sequence results obtained for hybridoma cell line ACE 8: the heavy chain coding gene sequence of the anti-APOE 4 point mutation monoclonal antibody is 1332bp long, and the sequence is shown as SEQ ID NO. 1; the light chain coding gene sequence of the anti-APOE 4 point mutation monoclonal antibody is 657bp long, and the sequence is shown as SEQ ID NO. 2. Deducing that the heavy chain encoded by the gene sequence consists of 444 amino acids according to the obtained gene sequence, wherein the sequence is shown as SEQ ID NO. 3; the light chain consists of 219 amino acids and has the sequence shown in SEQ ID No. 4.

Indirect ELISA detection method procedure:

1) Taking 96-well ELISA plates, adding 50 μl of immune complex with coating concentration diluted by coating solution into each well, namely 100ng of antigen, avoiding the use of edge wells as much as possible, reducing absorbance value to influence the result, and incubating at 4 ℃ overnight or at 37 ℃ for 2 hours;

2) Pouring off antigen, adding 100 μl of blocking solution (1%BSA,0.1M Kpi,0.1%Tween-20,0.02% merthiolate, pH 7) into each well, incubating overnight at 4deg.C or 37deg.C for 2 hr, pouring off blocking solution, washing the plate 3 times with washing solution (0.1M KPi,0.05%Tween-20, pH 7), and drying;

3) 50 μl of primary antibody is added per well, which is the cell supernatant if hybridoma cells are present, and a series of dilutions (blocking solution dilutions) are required if serum is present from the immunized animal, typically at an initial dilution of 1:499, followed by 2-fold serial dilutions, with 2 wells for negative, blank and positive controls, and incubation at 37deg.C for 1 hour; washing the plate for 3 times, and beating to dry;

4) Add 50 μl of 1 per well: 1999 dilution (blocking solution dilution) HRP-labeled secondary antibody, 37 ℃ incubation for 45min; washing the plate for 3 times, and beating to dry;

5) Mu.l TMB substrate (must be ready-to-use) was added to each well and incubated for 5-20 minutes at room temperature.

6) 100 μl of stop solution (0.5M oxalic acid) per well was added and the absorbance at 450nm was read on a microplate reader.

Immunofluorescence technical procedure:

1. before operation, the ultra-clean bench irradiates for 20 minutes by ultraviolet rays, and can perform subsequent operation after a fan is started for 5 minutes;

2. the primary rat hippocampal nerve cells/human brain glioma U87 cells (all purchased from An Nuolun (Wohan) biotechnology Co., ltd.) were digested with cell digestive juice, and gently beaten to prepare single cell suspension with a cell concentration of 4×10 ⁵ Inoculating sterile 96-well cell culture plates at a volume of 100 μl/well;

3. constructing a full-length expression plasmid containing APOE4 mutation sites of APOE4-112Arg and APOE4-112Cys protein pcDNA, transfecting primary rat hippocampal nerve cells/human brain glioma U87 cells with the plasmid, and simultaneously setting a normal cell blank control (adding 100 mu l of cell growth solution); and negative control 6 wells.

4. Placing CO at 37 DEG C ₂ Culturing in incubator; daily observation of cellsThe results were recorded and observed for 5-7 days.

5. The liquid in the 96-well plate of primary rat hippocampal nerve cells/human brain glioma U87 cells transfected with plasmids is discarded (negative wells are sucked first and positive wells are sucked later), the plate is washed 2 times with PBS with pH of 7.4, 150 μl of each well is washed, and the 96-well plate is placed on absorbent paper to be beaten after the plate washing is finished, so that no liquid remains in the wells.

6. After the plate washing was completed, 80% cold acetone was added to the 96-well plate, 150. Mu.l each well was allowed to stand at-20℃for 12 minutes.

7. After the fixation, the fixation fluid in the 96-well plate was removed, and the plate was washed once with PBS at pH7.4, 150. Mu.l per well. After the plate washing is finished, the 96-well plate is placed on absorbent paper to be beaten, so that no residual liquid exists in the well.

8. After the plate washing is finished, the 96-well plate can be stored for a long time at the temperature of minus 20 ℃.

9. The 96-well plate was charged with 3% BSA, 150 μl per well, and the wells were left at 37deg.C for 30 minutes and gently dried.

10. APOE4 point mutant mab was diluted with 1% bsa at a dilution ratio of 1:1000 times.

11. APOE4 point mutation monoclonal antibody is added into a 96-well plate, 50 mu l of each well is placed in a 37 ℃ incubator for 1 hour

12. Plates were washed 3 times with PBST, 150ml per well, 3-5 minutes apart.

13. Dilution of FITC-labeled goat anti-mouse IgG secondary antibody at a dilution ratio of 1:100 times (light-shading for secondary antibody)

14. The diluted secondary antibodies were added to the 96-well plate, 50. Mu.l per well, and left at 37℃for 1 hour.

15. The plates were washed 4 times with PBST, 150 μl each well, 3-5 minutes apart, and finally 1 time with pure water, 150 μl each well, and after washing, the 96-well plates were dried by pipetting.

16. The 96-well plate was blocked by adding 80% glycerol, 50 μl per well.

17. The results were observed.

The reagents used were as follows:

FITC-labeled goat anti-mouse IgG secondary antibody, product of KPL company, USA, imported BSA.

Acetone, glycerol, tween-80, KH2PO4, naCl, KCl and Na2HPO4.12H2O are all domestic analytical pure.

Preparation of wash solution PBST (PBS of pH7.4,0.05% Tween-20);

preparing a cell fixing solution: 80mL of acetone and 20mL of ultrapure water are mixed uniformly, and the mixture is preserved in a 200mL reagent bottle at the temperature of minus 20 ℃.

Example IV

Double-sandwich ELISA kit

An ELISA kit to test ApoE4 comprising:

the method comprises the steps of coating a solid phase carrier containing a capture antibody, a sample diluent, a standard substance protein, an enzyme-labeled primary antibody panApoE, a chromogenic substrate and a stop solution; the capture antibody was the antibody prepared in example two.

The capture antibody coating process is as follows:

(1) Taking a 96-well ELISA plate, adding 100 mu L of 0.05M carbonic acid buffer solution with the pH of 9.6 for dilution to obtain 10 mu g/L antibody solution, and incubating at 4 ℃ overnight or 37 ℃ for 2 hours to leave a plurality of blank holes;

(2) Pouring out the antibody solution, adding 100 μl of blocking solution (1%BSA,0.1M Kpi,0.1%Tween-20,0.02% merthiolate, pH 7) into each well, incubating overnight at 4deg.C or 37deg.C for 2 hr, pouring out the blocking solution, washing the plate 3 times with washing solution (0.1M KPi,0.05%Tween-20, pH 7), and drying by beating;

(3) Vacuum drying, packaging to obtain solid phase carrier containing capture antibody, and storing at 4deg.C.

The standard protein is ApoE4 standard lyophilized powder (general biological (Wuhan) technology Co., ltd.).

The sample diluent is 0.01mol/L phosphate buffer solution, contains 1% of BSA, 0.05% of Tween-20 and 1mg/L gentamicin by mass percent, and is packaged into 15 ml/bottle.

The termination liquid is H of 2mol/L ₂ SO ₄ The package was 5 ml/tube.

The enzyme-labeled primary antibody is horseradish peroxidase-labeled primary antibody panApoE.

The preparation process is as follows:

horseradish peroxidase is weighed and dissolved in deionized waterAdding newly prepared 0.1MNaIO into the son water ₄ Stirring at room temperature in dark until the solution is brown green;

the solution was dialyzed overnight at low temperature in 1M sodium acetate buffer at pH 4.4.

Adding carbonate buffer solution into the solution until the pH value is 9-9.5, adding a pan ApoE, and stirring in a dark place;

adding 0.1ml of newly prepared 4mg/L NaBH ₄ The solution is evenly mixed and placed at 4 ℃ for reaction for 2 hours.

The above solution was packed in a dialysis bag and dialyzed against 0.15M PBS pH7.4 at 4℃overnight.

10000g is centrifugated for 15min, the precipitated protein is discarded, and the supernatant is the enzyme-labeled primary antibody pan ApoE.

Further, the color-developing agent is TMB substrate color-developing agent, and the dosage is 10 ml/tube.

Example five

Fourth example method of Using the kit

1. Preparation of sample to be tested

Collecting human blood, placing into a centrifuge tube, centrifuging at 2500rpm for 10min after blood is coagulated to obtain serum sample, and preserving at-20deg.C to avoid repeated freezing and thawing.

Preparation of standard solutions

10 sample tubes were removed.

And preparing standard substance solutions with different concentrations by using the sample diluent and the freeze-dried powder of the ApoE4 standard substance.

3. Test procedure

(1) Adding a sample to be tested into a sample diluent to dilute by 500 times, taking 100 mu l of the sample to be tested, adding the sample diluent into an ELISA plate coated with an antibody, adding 100 mu l of the sample diluent into some blank holes, and adding a standard solution into other blank holes;

(2) Incubating for 2 hours at 37 ℃;

(3) Washing the plate for 4 times, and then beating to dry;

(4) Adding 100 μl of enzyme-labeled antibody or enzyme-labeled primary antibody pan ApoE solution diluted 2000 times with sample diluent, incubating for 2 hours at 37deg.C, washing the plate for 4 times, and drying;

(5) 100 μl of TMB substrate developer was added to each well, incubating for 15-20 min at room temperature. 100 μl of stop solution per well was added and the absorbance at 450nm was read on a microplate reader.

(6) Solving a curve of concentration and absorbance of the standard solution;

(7) And (5) taking the degree of the sample to be tested into a curve, and calculating to obtain a test result.

Example six

Detection accuracy test

Standard weighing ApoE4 standard substance freeze-dried powder, adding sample diluent to prepare three groups of standard substance solutions with different concentrations, testing each standard substance solution for three times according to the fifth test process of the embodiment to obtain a detection concentration, and calculating to obtain the difference between the detection concentration and the nominal concentration, wherein the calculation formula is as follows: ((detection concentration-nominal concentration)/nominal concentration) 100%, the detection result is 5.4%,7.6% and 4.8%, and the result shows that the test accuracy of the kit is high.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> Wuhan Tiande Biotech Co., ltd

<120> ELISA kit for testing ApoE4 protein content

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1332

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

caggtgcagc tggtgcagag cggcgccgag gtgaagcgcc ccggcctgag catccgcatc 60

agctgccgcc tgagcggcta caccttcacc aactaccaga tgcactgggt gcgccaggcc 120

cccggcaacc gcctggagtg gatgggcacc atctaccccg gcaacgacga caccagctac 180

aacaaccgct tccgcgagaa gatcaccgtg accctggaga ccagcctgag caccctgtac 240

atggacgcca gcagcgccaa gagcgacgac accgccgtgt actactgcgc caagggcggc 300

taccgcgcca tggagtactt cggcaacggc accgccatca ccatcagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tgcagccgca gcaccagcga gagcaccgcc 420

gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480

ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540

agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcaccaagac ctacacctgc 600

aacgtggacc acaagcccag caacaccaag gtggacaagc gcgtggagag caagtacggc 660

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 720

ccccccaagc ccaaggacac cctgatgatc agccgcaccc ccgaggtgac ctgcgtggtg 780

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 840

gtgcacaacg ccaagaccaa gccccgcgag gagcagttca acagcaccta ccgcgtggtg 900

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 1020

cgcgagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 1080

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1200

ttcttcctgt acagccgcct gaccgtggac aagagccgct ggcaggaggg caacgtgttc 1260

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1320

agcctgggca ag 1332

<210> 2

<211> 657

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gaggtgatca tgaccaacag ccccgccagc gcccccatca cccccggcga gcccctgagc 60

atcagctgca agagcagcaa cagcgtgatc tacagcaacg gcaacaccta cctgggctgg 120

tacctgcaga agcccggcaa cagccccaac gccgccgtgt accgcgtgag caaccgcttc 180

agcggcgtgc ccgaccgctt cagcggcagc ggcagcggca ccgacttcac cctgaaggtg 240

agccgcgtgg aggccgagga cgtgggcatc tactactgct ggaacggcag ccacatcccc 300

tacacctggg gccagggcac caaggccgag atcaagcgca ccgtggccgc ccccagcgtg 360

ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg 540

agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600

gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgcgg cgagtgc 657

<210> 3

<211> 444

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Gly Val Gly Leu Val Gly Ser Gly Ala Gly Val Leu Ala Pro Gly Leu

1 5 10 15

Ser Ile Ala Ile Ser Cys Ala Leu Ser Gly Thr Thr Pro Thr Ala Thr

20 25 30

Gly Met His Thr Val Ala Gly Ala Pro Gly Ala Ala Leu Gly Thr Met

35 40 45

Gly Thr Ile Thr Pro Gly Ala Ala Ala Thr Ser Thr Ala Ala Ala Pro

50 55 60

Ala Gly Leu Ile Thr Val Thr Leu Gly Thr Ser Leu Ser Thr Leu Thr

65 70 75 80

Met Ala Ala Ser Ser Ala Leu Ser Ala Ala Thr Ala Val Thr Thr Cys

85 90 95

Ala Leu Gly Gly Thr Ala Ala Met Gly Thr Pro Gly Ala Gly Thr Ala

100 105 110

Ile Thr Ile Ser Ser Ala Ser Thr Leu Gly Pro Ser Val Pro Pro Leu

115 120 125

Ala Pro Cys Ser Ala Ser Thr Ser Gly Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Leu Ala Thr Pro Pro Gly Pro Val Thr Val Ser Thr Ala Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Pro Pro Ala Val Leu Gly Ser

165 170 175

Ser Gly Leu Thr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Leu Thr Thr Thr Cys Ala Val Ala His Leu Pro Ser Ala

195 200 205

Thr Leu Val Ala Leu Ala Val Gly Ser Leu Thr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Gly Pro Leu Gly Gly Pro Ser Val Pro Leu Pro

225 230 235 240

Pro Pro Leu Pro Leu Ala Thr Leu Met Ile Ser Ala Thr Pro Gly Val

245 250 255

Thr Cys Val Val Val Ala Val Ser Gly Gly Ala Pro Gly Val Gly Pro

260 265 270

Ala Thr Thr Val Ala Gly Val Gly Val His Ala Ala Leu Thr Leu Pro

275 280 285

Ala Gly Gly Gly Pro Ala Ser Thr Thr Ala Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gly Ala Thr Leu Ala Gly Leu Gly Thr Leu Cys Leu Val

305 310 315 320

Ser Ala Leu Gly Leu Pro Ser Ser Ile Gly Leu Thr Ile Ser Leu Ala

325 330 335

Leu Gly Gly Pro Ala Gly Pro Gly Val Thr Thr Leu Pro Pro Ser Gly

340 345 350

Gly Gly Met Thr Leu Ala Gly Val Ser Leu Thr Cys Leu Val Leu Gly

355 360 365

Pro Thr Pro Ser Ala Ile Ala Val Gly Thr Gly Ser Ala Gly Gly Pro

370 375 380

Gly Ala Ala Thr Leu Thr Thr Pro Pro Val Leu Ala Ser Ala Gly Ser

385 390 395 400

Pro Pro Leu Thr Ser Ala Leu Thr Val Ala Leu Ser Ala Thr Gly Gly

405 410 415

Gly Ala Val Pro Ser Cys Ser Val Met His Gly Ala Leu His Ala His

420 425 430

Thr Thr Gly Leu Ser Leu Ser Leu Ser Leu Gly Leu

435 440

<210> 4

<211> 219

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Gly Val Ile Met Thr Ala Ser Pro Ala Ser Ala Pro Ile Thr Pro Gly

1 5 10 15

Gly Pro Leu Ser Ile Ser Cys Leu Ser Ser Ala Ser Val Ile Thr Ser

20 25 30

Ala Gly Ala Thr Thr Leu Gly Thr Thr Leu Gly Leu Pro Gly Ala Ser

35 40 45

Pro Ala Ala Ala Val Thr Ala Val Ser Ala Ala Pro Ser Gly Val Pro

50 55 60

Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Val

65 70 75 80

Ser Ala Val Gly Ala Gly Ala Val Gly Ile Thr Thr Cys Thr Ala Gly

85 90 95

Ser His Ile Pro Thr Thr Thr Gly Gly Gly Thr Leu Ala Gly Ile Leu

100 105 110

Ala Thr Val Ala Ala Pro Ser Val Pro Ile Pro Pro Pro Ser Ala Gly

115 120 125

Gly Leu Leu Ser Gly Thr Ala Ser Val Val Cys Leu Leu Ala Ala Pro

130 135 140

Thr Pro Ala Gly Ala Leu Val Gly Thr Leu Val Ala Ala Ala Leu Gly

145 150 155 160

Ser Gly Ala Ser Gly Gly Ser Val Thr Gly Gly Ala Ser Leu Ala Ser

165 170 175

Thr Thr Ser Leu Ser Ser Thr Leu Thr Leu Ser Leu Ala Ala Thr Gly

180 185 190

Leu His Leu Val Thr Ala Cys Gly Val Thr His Gly Gly Leu Ser Ser

195 200 205

Pro Val Thr Leu Ser Pro Ala Ala Gly Gly Cys

210 215

<210> 5

<211> 16

<212> PRT

<213> human body (homosapiens)

<400> 5

Gly Ala Val Ala Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala

1 5 10 15

<210> 6

<211> 897

<212> DNA

<213> human ApoE2 (homosapiens)

<400> 6

aaggtggagc aggccgtgga gacagagcct gagcctgagc tgagacagca gaccgagtgg 60

cagagcggcc agaggtggga gctggccctg ggaagatttt gggattacct gagatgggtg 120

cagaccctgt ccgagcaggt gcaggaggag ctgctgtcca gccaggtgac ccaggagctg 180

agggccttga tggacgagac aatgaaggag ctgaaggctt ataagtccga gctggaggag 240

cagctgaccc ccgtggccga ggagacaaga gccaggctgt ccaaggagct gcaagccgcc 300

caggccaggc tgggagctga catggaggat gtgtgcggca ggctggtgca gtacagaggc 360

gaggtgcagg ccatgctggg ccagtccacc gaggagctga gggtgagact ggcctcccac 420

ctgaggaagc tgaggaagag gctgctgaga gacgccgacg atctgcaaaa gtgcctggcc 480

gtgtaccagg ccggcgccag agagggagcc gagagaggac tgtccgccat cagagagaga 540

ctgggacctc tggtggagca gggcagagtg agggccgcca ccgtgggaag cctggctgga 600

cagcctctgc aagagagggc tcaagcctgg ggcgagagac tgagagccag gatggaggag 660

atgggcagca ggaccaggga cagactggac gaggtgaagg agcaggtggc cgaggtgaga 720

gccaagctgg aggagcaagc ccagcagatc aggctgcaag ccgaggcttt tcaggccaga 780

ctgaagagct ggttcgagcc tctggtggaa gacatgcaga gacagtgggc cggcctggtg 840

gagaaggtgc aggccgctgt gggcacaagc gccgctccag tgcctagcga taaccac 897

<210> 7

<211> 897

<212> DNA

<213> human ApoE3 (homosapiens)

<400> 7

aaggtggagc aggccgtgga gacagagcct gagcctgagc tgaggcagca gaccgagtgg 60

cagtccggcc agaggtggga gctggccctg ggaagattct gggattacct gaggtgggtg 120

cagaccctgt ccgagcaggt gcaggaggag ctgctgagca gccaggtgac ccaggagctg 180

agagccctga tggacgagac aatgaaggag ctgaaggctt ataagagcga gctggaggag 240

cagctgacac ccgtggccga ggagacaagg gccagactga gcaaggagct gcaagccgcc 300

caggccagac tgggcgctga catggaggac gtgtgcggca gactggtgca gtacaggggc 360

gaggtgcagg ccatgctggg ccagagcaca gaggagctga gagtgagact ggcctcccac 420

ctgaggaagc tgaggaagag gctgctgagg gatgccgatg acctgcaaaa gagactggcc 480

gtgtaccagg ccggcgccag ggaaggagcc gagagaggac tgtccgccat cagagagagg 540

ctgggacctc tggtggagca gggcagggtg agggccgcta ccgtgggaag cctggccgga 600

cagcccctgc aagagagagc ccaggcttgg ggcgagagac tgagagccag gatggaggag 660

atgggctcca ggaccaggga caggctggat gaggtgaagg agcaggtggc cgaggtgaga 720

gccaagctgg aggagcaagc ccagcagatc aggctgcaag ccgaggcttt tcaggccagg 780

ctgaagagct ggtttgagcc cctggtggag gatatgcaga gacagtgggc cggcctggtg 840

gagaaggtgc aggccgctgt gggcaccagc gccgctcctg ttcctagcga caatcac 897

<210> 8

<211> 897

<212> DNA

<213> human ApoE4 (homosapiens)

<400> 8

aaggtggagc aggccgtgga gacagagccc gagcctgagc tgagacagca gaccgagtgg 60

cagtccggcc agagatggga gctggccctg ggcagattct gggactacct gaggtgggtg 120

cagacactgt ccgagcaggt gcaggaggag ctgctgtcca gccaggtgac ccaggagctg 180

agggccttga tggatgagac aatgaaggag ctgaaggctt ataagtccga gctggaggag 240

cagctgacac ccgtggccga ggagacaaga gccagactga gcaaggagct gcaagccgcc 300

caggccagac tgggcgctga tatggaggat gtgagaggca gactggtgca gtacagaggc 360

gaggtgcagg ccatgctggg ccagagcaca gaggagctga gagtgaggct ggcctcccac 420

ctgaggaagc tgagaaagag actgctgagg gatgccgatg acctgcaaaa gaggctggcc 480

gtgtaccagg ccggcgccag agagggagcc gagagaggac tgtccgccat cagggagaga 540

ctgggacctc tggtggagca gggcagggtg agagccgcca ccgtgggaag cctggccgga 600

caacctctgc aagagagggc tcaagcctgg ggcgagaggc tgagggccag aatggaggag 660

atgggctcca ggacaagaga tagactggac gaggtgaagg agcaggtggc cgaggtgagg 720

gccaagctgg aggagcaagc ccagcagatc agactgcaag ccgaggcttt tcaggccagg 780

ctgaagtcct ggttcgagcc cctggtggag gacatgcaga ggcagtgggc cggcctggtg 840

gagaaggtgc aggccgctgt gggcacatcc gccgctcctg tgccctccga caatcac 897

<210> 9

<211> 299

<212> PRT

<213> human ApoE2 (homosapiens)

<400> 9

Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly

1 5 10 15

Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala

20 25 30

Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly

35 40 45

Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met

50 55 60

Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly

65 70 75 80

Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly

85 90 95

Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Cys

100 105 110

Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly

115 120 125

Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu

130 135 140

Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Cys Leu Ala

145 150 155 160

Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala

165 170 175

Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly

195 200 205

Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala

210 215 220

Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala

225 230 235 240

Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala

245 250 255

Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met

260 265 270

Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly

275 280 285

Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His

290 295

<210> 10

<211> 299

<212> PRT

<213> human ApoE3 (homosapiens)

<400> 10

Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly

1 5 10 15

Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala

20 25 30

Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly

35 40 45

Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met

50 55 60

Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly

65 70 75 80

Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly

85 90 95

Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Cys

100 105 110

Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly

115 120 125

Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu

130 135 140

Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Ala Leu Ala

145 150 155 160

Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala

165 170 175

Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly

195 200 205

Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala

210 215 220

Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala

225 230 235 240

Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala

245 250 255

Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met

260 265 270

Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly

275 280 285

Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His

290 295

<210> 11

<211> 299

<212> PRT

<213> human ApoE4 (homosapiens)

<400> 11

Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly

1 5 10 15

Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala

20 25 30

Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly

35 40 45

Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met

50 55 60

Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly

65 70 75 80

Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly

85 90 95

Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Ala

100 105 110

Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly

115 120 125

Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu

130 135 140

Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Ala Leu Ala

145 150 155 160

Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala

165 170 175

Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala

180 185 190

Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly

195 200 205

Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala

210 215 220

Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala

225 230 235 240

Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala

245 250 255

Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met

260 265 270

Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly

275 280 285

Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His

290 295

Claims

1. An ELISA kit to test ApoE4 protein content, characterized in that the ELISA kit to test ApoE4 comprises:

the kit comprises a solid phase carrier containing a capture antibody, a sample diluent, a standard protein, an enzyme-labeled primary anti-pan ApoE, a color developing agent and a stop solution;

wherein the capture antibody is an anti-APOE 4 point mutation monoclonal antibody, and the heavy chain amino acid sequence of the anti-APOE 4 point mutation monoclonal antibody is shown in SEQ ID NO:3, the light chain amino acid sequence of the anti-APOE 4 point mutation monoclonal antibody is shown as SEQ ID NO: 4.

2. The ELISA kit for testing the protein content of ApoE4 according to claim 1, wherein the anti-APOE 4 point mutation monoclonal antibody is prepared by a hybridoma cell strain, wherein the hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is: cctccc NO: C2020121.

3. the ELISA kit for testing the content of the ApoE4 protein according to claim 1, wherein the standard protein is ApoE4 standard lyophilized powder.

4. The ELISA kit for testing the protein content of ApoE4 according to claim 1, wherein the sample diluent is 0.01mol/L phosphate buffer solution and contains 1% of BSA, 0.05% of Tween-20 and 1mg/L gentamicin by mass percent.

5. The ELISA kit for testing the protein content of ApoE4 according to claim 1, wherein the stop solution is 2mol/L H ₂ SO ₄ 。

6. The ELISA kit of claim 1 wherein the enzyme-labeled primary antibody pan ApoE is horseradish peroxidase-labeled murine monoclonal antibody pan ApoE.

7. The ELISA kit of claim 1 wherein the chromogenic reagent is TMB substrate chromogenic reagent.