CN112540179A

CN112540179A - ELISA kit for testing ApoE4 protein content

Info

Publication number: CN112540179A
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: 2021-03-23
Anticipated expiration: 2040-08-06
Also published as: CN112540179B

Abstract

The invention provides an ELISA kit for testing ApoE4 protein content, which comprises: comprises a solid phase carrier containing a capture antibody, a sample diluent, a standard protein, enzyme-labeled anti-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 following beneficial effects: the double-sandwich monoclonal antibody ELISA kit provided by the invention can specifically identify APOE4 point mutant protein, and is screened by two screening modes of APOE4-Arg and APOE4-Cys epitope peptide synthesized by a coating solid phase method and APOE4-Arg and APOE4-Cys protein produced by coating genetic engineering cells, so that the kit has good sensitivity, specificity and accuracy.

Description

ELISA kit for testing ApoE4 protein content

Technical Field

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

Background

Alzheimer's Disease (AD), also known as senile dementia, is an age-related neurodegenerative disease whose main clinical manifestations are memory dysfunction and changes in behavior. As a progressive disease, the most important physiological feature of AD is that areas of the brain undergo progressive dysfunction, neuronal loss, and loss of synapses. The Bateman et al study demonstrated that the brain of patients began to develop changes gradually 10 to 20 years before the onset of AD. These functional changes are often difficult to reverse, which also leads to difficulties with current AD treatments. Therefore, detection of early AD may be the most effective means for preventing and treating AD.

The detection of blood biomarkers is the preferred means for the detection of numerous diseases due to its advantages of being inexpensive, easy to operate, and non-invasive. In AD, a plurality of proteins are proved to have obvious changes in the plasma of AD patients, such as BDNF, AGT, IGFBP-2, OPN and the like, and the detection of the protein content in the plasma is suggested to be a valuable mode for early diagnosis of AD.

While apolipoprotein e (apoe) has been shown to be associated with the development of AD. The apolipoprotein (Apo) E gene is divided into three allelic forms, epsilon 2, epsilon 3 and epsilon 4, which respectively encode three isoforms of ApoE2, ApoE3 and ApoE 4. The three different subtypes of ApoE differ only in their primary structure 112 and the amino acid at position 158, i.e. ApoE2(112Cys,158Cys), ApoE3(112Cys,158Arg), ApoE4(112Arg,158 Arg). Among them, high expression of ApoE epsilon 4 gene has been recognized as one of the largest risk factors for AD. Immunohistochemical analysis of Sporadic AD (SAD) and late Familial AD (FAD), which account for the majority of the disease, at Duke university in 1990 revealed the presence of ApoE4 in senile plaques and neurofibrillary tangles. Studies have shown that both sporadic and familial late-onset AD is associated with epsilon 4. It is found that the carrier of heterozygote epsilon 4 has 3 times higher probability of having late-onset AD than the non-carrier, and the carrier of homozygote epsilon 4 has 10-20 times higher probability of having AD than the non-carrier. Carwfold et al found that the ε 4 gene was correlated with patient age and onset age. In early diagnosis, however, an individual carrying ApoE4 would have at least 5-10% confidence added to the diagnosis of AD, particularly for differential diagnosis of different types of dementia. Meanwhile, if the patient has AD-related behavioral phenotype and carries ApoE4, the accuracy of AD diagnosis can reach 94% -98%. These all suggest that: detection of the genotype of ApoE4 is probably one of the most valuable indicators for early diagnosis of AD.

However, currently, the detection of ApoE genotypes 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 guarantee. According to the quality evaluation report results of the second generation sequencing laboratories of the national tumors at the end of 2015, 55% of the sequencing results of the laboratories are unqualified, and 22% of the laboratory quality evaluation is 0 point. These result in ApoE4 genotype tests that are difficult to generalize for AD diagnosis.

And changes in genotype will result in changes in protein expression. According to literature reports, the difference of ApoE allelic genes can cause the serum apoE inhibiting concentration of AD patients to change and cause abnormal blood lipid metabolism of the AD patients; the serum ApoE4 concentration of AD patients is obviously higher than that of normal people. It is suggested that detecting the content of ApoE4 protein in serum may have very important significance in early diagnosis of AD. At present, no ApoE4 protein detection kit is on the market at home and abroad. Meanwhile, the protein kit has the advantages of high accuracy and low cost in detection. Therefore, researchers think that the development of the kit for detecting the serum ApoE4 protein can provide strong evidence for early AD diagnosis, and the kit has certain practical significance and economic benefit.

The application of the monoclonal antibody technology in disease diagnosis and drug development is a research hotspot in the field of medicine at present, and provides a new way for treating various diseases.

Currently, ApoE4 monoclonal antibody for detection cannot completely distinguish ApoE2 from ApoE3, so ApoE4 monoclonal antibody has weak specificity for ApoE4 detection, and further, no ApoE4 reagent detection product with strong specificity can be developed.

Disclosure of Invention

In order to develop a rapid, sensitive and strong-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, except that the ELISA kit for testing ApoE4 comprises:

the kit comprises a solid phase carrier containing a capture antibody, a sample diluent, a standard protein, enzyme-labeled primary anti-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 shown as SEQ NO:3, the light chain amino acid sequence of the anti-APOE 4 point mutation monoclonal antibody is shown as SEQ NO:4, respectively.

Further, the APOE4 point mutation resistant monoclonal antibody is prepared from a hybridoma cell strain which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of: CCTCC NO: 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 in percentage by mass.

Further, the stop solution is 2mol/L of H₂SO₄。

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

Further, the color developing agent is a 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 APOE4 point mutant protein, and is screened by two screening modes of APOE4-Arg and APOE4-Cys protein synthesized by a coating solid phase method and APOE4-Arg and APOE4-Cys protein produced by coating genetic engineering cells, so that the kit has good sensitivity, specificity and accuracy.

Drawings

FIG. 1 is a SDS-PAEG electrophoretogram of purified antibody, wherein MW: represents a molecular weight; lane 1 shows ACE8 denatured without boiling; lane 2; indicating that ACE8 was denatured by boiling;

FIG. 2 shows Western blot analysis of transfected HeLa cells expressing extracts of full-length human ApoE2(hApoE2), ApoE3(hApoE3) or ApoE4(hApoE4), wherein the primary antibody used in the above figure is a monoclonal antibody produced by hybridoma ACE8 of the present invention, and the primary antibody used in the following figure is a panApoE antibody;

FIG. 3 is a graph showing the results of immunofluorescence analysis of primary rat hippocampal neural cells using ApoE4(ACE8) mouse mAb murine mAb;

FIG. 4 shows immunofluorescent cell staining of human brain glioma U87 cells with ApoE4(ACE8) mouse mAb murine mAb;

FIG. 5 is a graph of the results of immunohistochemical analysis of paraffin-embedded human skin using ApoE4(ACE8) mouse mAb murine mAb, where A is a control peptide and B is an antigen-specific peptide;

FIG. 6 is a graph of the results of immunohistochemical analysis of paraffin-embedded human kidney tissue sections using ApoE4(ACE8) mouse mAb murine mAb;

FIG. 7 is a graph of the results of immunohistochemical analysis of paraffin-embedded human spleen tissue sections using ApoE4(ACE8) mouse mAb murine mAb;

FIG. 8 is a graph of the results of immunohistochemical analysis of paraffin-embedded human colon tissue sections using ApoE4(ACE8) mouse mAb murine mAb.

Detailed Description

The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention.

Example 1 immune Complex preparation

(1) The 16 peptide of APOE4 protein (the amino acid sequence at position 109-124 of APOE4 protein: EDVRGRLVQYRGEVQA, shown in SEQ ID NO. 5) was synthesized by a solid phase method on a peptide synthesizer (431A) of ABI, USA, using Fmoc (9-fluorenylmethyloxycarbonyl) protocol, and the synthesis steps were performed according to the manual of the peptide synthesis operation of ABI. Purifying by high performance liquid chromatography, identifying the sequence, and preparing the epitope peptide of the APOE4 protein by mass spectrometry.

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

The method can be realized by adopting common reagents, instruments and the like in the field.

EXAMPLE 2 monoclonal antibody preparation

(1) Animal immunization

Six female Balb/c pure mice of 6-8 weeks old were used and five immunization injections were performed according to the following immunization method.

First immunization: on day 0, each mouse groin was injected subcutaneously with 0.2ml of an emulsion of the immune complex solution and freund's complete adjuvant at 1:1, the emulsion contains 30-50 mu g of immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

And (3) second immunization: on day 21, 0.2ml of emulsion, which is an immune complex solution and incomplete freund's adjuvant, was injected subcutaneously into the groin of each mouse using a 2ml syringe at a rate of 1:1, the emulsion contains 30-50 mu g of immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

And (3) third immunization: on day 42, each mouse groin was injected subcutaneously with 0.2ml of an emulsion of the immune complex solution and Freund's incomplete adjuvant at 1:1, the emulsion contains 30-50 mu g of immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

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

And (4) fifth immunization: on day 78, 0.2ml of emulsion was injected into the tail vein of each mouse using a 2ml syringe, wherein the emulsion was an immune complex solution and Freund's incomplete adjuvant at a ratio of 1:1, the emulsion contains 30-50 mu g of immune complex, and the immune complex solution is obtained by diluting the immune complex with 1 XPBS.

The immune serum titer was tested by indirect ELISA on day 1, day 52 and day 73, respectively, wherein the titer reached 128K or more on day 73, the tail vein of the mice was subjected to shock immunization on day 78, and the spleen B cells of the mice were taken 3 days later (day 81) for fusion.

(2) Preparation of hybridoma cells

Preparation of mouse myeloma cell SP 2/0: culturing SP2/0 myeloma cell line from BALB/C mouse in 10% FBS-DMEM medium, passaging in 5% CO₂The culture was carried out in a 37 ℃ incubator saturated in humidity. The day before fusion was passaged to ensure that cells entered logarithmic growth phase at the time of fusion.

Preparation of feeder cells: macrophages in the abdominal cavity of the mice were taken as feeder cells. Normal BALB/c mice were collected by removing the eyeballs, and the serum was isolated as a negative control serum for antibody detection. Meanwhile, the mouse is killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on a dissecting table plate, then left abdominal skin is lifted, spleen can be seen, forceps are cut for ophthalmology, peritoneum is cut by a sterile surgical scissors in a super clean bench, the spleen is taken out and placed in a flat dish, and surrounding connective tissues are carefully stripped. The spleen was squeezed with a plunger of a syringe to disperse the cells, and the cells were purged several times with 10ml of an incomplete medium to prepare a single cell suspension. Transferring into a50 ml centrifuge tube, centrifuging for 5-10 minutes at 1000r/min, centrifuging and washing for 1-2 times by using an incomplete culture medium, then suspending the cells in 10ml incomplete culture medium, mixing uniformly, taking the suspension, and adding a phenol blue staining solution for counting the living cells for later use. Usually 1X 10 can be obtained per mouse⁸-2.5×10⁸Spleen cells, the cell suspension was added to a 96-well plate at 0.1ml per well (equivalent to 2 drops), and then placed at 37 ℃ in 4% CO₂Cultured in an incubator.

Preparation of immune spleen cells: pulling neck or CO of immunized Balb/c mice₂Killing, soaking in 75% ethanol solution for 5min, aseptically opening abdomen in a super clean bench, taking out spleen, and washing once with 5ml of RPMI1640 incomplete culture solution; placing a stainless steel net in a plate containing 20ml of incomplete culture solution, moving the spleen onto a screen, slightly grinding the spleen by using an inner core of a syringe, sucking the culture medium solution in the plate, slightly washing the stainless steel screen, and enabling all spleen cells to enter the solution through meshes; transferring the above spleen cell solution toAdding 15-20ml of incomplete culture solution into a50 ml centrifuge tube, and uniformly mixing; centrifuging at 1200r/min for 10min, and removing the supernatant; and centrifuging and washing the cell sediment once by using an incomplete culture solution and the same method, then resuspending, and staining trypan blue to count the living cells. The typical immune spleen cell volume is about 2 times of the normal spleen volume, and 1.0X 10 can be obtained per mouse⁸-2.5×10⁸And (4) spleen cells.

Cell fusion: taking myeloma cells SP2/0 in logarithmic phase, centrifuging for 5 minutes at 1000r/min, removing supernatant, re-suspending cell precipitates by using incomplete culture solution, uniformly mixing, dyeing trypan blue or counting cells, taking required cells, and washing for 2 times by using the incomplete culture solution; washing the prepared immune splenocytes for 2 times by using incomplete culture solution; adding myeloma cells and splenocytes into a same 50ml centrifugal tube according to the proportion of 1:10 or 1:5, supplementing incomplete culture solution to 30-40ml, and mixing well; centrifuging at 1200r/min for 10min, discarding the supernatant, and lightly flicking the bottom of the centrifuge tube to loosen the cell precipitate into uniform paste. Fusing at room temperature: the centrifuge tube was rotated uniformly with one hand and 45% PEG60001ml preheated at 37 ℃ was aspirated with a 1ml pipette with the other hand and added uniformly and slowly along the wall of the rotated centrifuge tube for about 60 seconds, then the cell suspension was immediately and gently aspirated into the pipette for about 30 seconds, left to stand for 30 seconds, then gently insufflated into the centrifuge tube for about 30 seconds, and the incomplete culture medium preheated to 37 ℃ was immediately added at volumes of 1, 2, 4, 8 and 10ml per minute (25 ml total) within five minutes to terminate its pro-cell fusion by diluting PEG. Centrifuging at 800r/min for 6 min, discarding the supernatant, adding appropriate amount of 20% FCS-RPMI1640 complete culture solution, suspending the fused cells gently, and blowing and sucking the cells without force. The complete medium was used in a 10ml block of 96 well cell culture plates.

The fused cell suspension was added to a 96-well cell culture plate plated with feeder cells at 100. mu.l/well, 37 ℃ with 5% CO₂Culturing in an incubator. Usually 4-6 96-well plates are added after fusion of 1 immunized mouse spleen cell.

(3) Screening and cloning of hybridoma cells

1/2 medium (HAT) was changed every 4 days, and HT-containing selective medium was used 10 days later. The fused hybridoma cells were cultured in selective medium containing HT for approximately two weeks. When the cell colony grows to a proper size (observed under a 10-fold objective lens, the cell clone size is preferably one field), the culture supernatant is sucked up, and after proper dilution, indirect ELISA is carried out to screen positive clones.

a. Preliminary screening

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

b. Secondary screening

Cloning a plasmid containing nucleotide sequences encoding APOE4-112Cys, i.e., ApoE2(112Cys,158Cys), ApoE3(112Cys,158Arg) and APOE4-112Arg, i.e., ApoE4(112Arg,158Arg), proteins (wherein the nucleotide sequence encoding ApoE2 is shown in SEQ ID NO: 6, the amino acid sequence of the encoded ApoE2 protein is shown in SEQ ID NO: 9, the nucleotide sequence encoding ApoE3 is shown in SEQ ID NO: 7, the amino acid sequence of the encoded ApoE2 protein is shown in SEQ ID NO: 10, the nucleotide sequence encoding ApoE4 is shown in SEQ ID NO: 8, and the amino acid sequence of the encoded ApoE2 protein is shown in SEQ ID NO: 11) onto pcDNA3.1(+) full-length expression and preparing the plasmid according to standard molecular biology methods, infecting HEK293 cells (purchased from ATCC, USA) with a transient transfection method, the cells are then lysed and the expressed protein purified.

The three proteins are coated, and the preliminarily screened positive cell strains are further screened by an indirect ELISA method, wherein the cell strains which have positive reaction on APOE4-112Arg proteins and have no reaction on APOE4-112Cys proteins are regarded as positive cell strains.

The selected positive cell lines were cloned by limiting dilution method. The single colony clones were selected and positive cell lines were further determined by the methods of a and b.

Preparation of hybridoma cell suspension: gently blowing off hybridoma cells detected as positive holes by ELISA, 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 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, 2 of a 96-well cell culture plate, where the concentration of hybridoma cells in the two rows was 10 per well. Adding equal volume of feeder cell suspension into the rest cell suspension, mixing uniformly, adding into the 3 rd and 4 th rows, and repeating the above steps until the concentration of hybridoma cells in the two rows is 5 per well, and the concentration of hybridoma cells in the 5 th and 6 th rows is 2.5 per well, and the concentration of hybridoma cells in the 7 th and 8 th rows is 1.25 per well. 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 plate was carefully aspirated, and 100. mu.L of complete medium was further added. The supernatants of the wells were tested by indirect ELISA, high titer cell culture wells were selected for recloning and monoclonal wells were selected as soon as possible. Until the monoclonal wells of two successive cloning tests were all positive.

6 hybridoma cell lines capable of stably secreting APOE4 point mutation-resistant monoclonal antibodies are obtained by cell fusion and are shown in Table 1 below. The antibody secreted by the hybridoma cell strain ACE8 has the best effect when being used for detecting APOE4 protein, and is preserved in China center for type culture Collection (CCTCC NO: C2020121) at 7-22.2020.

TABLE 1 hybridoma cell lines stably secreting monoclonal antibodies against ApoE4 protein

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

The titer of the supernatant of the hybridoma cell line was measured by indirect ELISA, and the supernatant was diluted 2-fold stepwise from 3 rd to 10 th wells with 0.01MpH7.2 PBS buffer. The 1 st hole uses the serum of the mouse diluted to 100 times during fusion as a positive control, the 2 nd hole uses the RPMI1640 complete culture solution as a 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 detection system is effective, when the OD value is more than or equal to 2 times the OD value of the negative control, the detection system is positive, otherwise, the detection system is negative. The dilution ratio corresponding to the lowest positive well is the titer of the culture supernatant of the hybridoma cell strain, which can reach 4096K (K, 1000,4096K represents 4096000), as shown in Table 2 below.

TABLE 2 results of supernatant titer of hybridoma cell lines

(4) Ascites production and purification of monoclonal antibodies

Inducing ascites: 1 week before inoculating hybridoma cells, 0.3-0.5 ml/liquid paraffin is inoculated to abdominal cavity of mouse to sensitize mouse. 7-10 days later, hybridoma cells diluted with PBS or serum-free medium were inoculated into the abdominal cavity of mice at 3X 10/mouse⁵0.5 ml. The ascites of the mice is observed every day after inoculation, if the abdomen is obviously enlarged and the skin is tense when the mice are touched by hands, the mice can be killed by pulling the neckThe ascites is sucked into a 15ml centrifuge tube by a dropper, and 1-5ml ascites can be obtained from one mouse. Centrifuging the collected ascites to obtain supernatant, and storing the small sample in a refrigerator below-20 deg.C for use.

Purification of monoclonal antibodies: the collected ascites is purified by an ammonium sulfate precipitation method. The purity of the antibody was verified and quantified by SDS-PAGE, and the results are shown in FIG. 1.

The specific purification steps are as follows: 1) adding 1 part of ascites into 2 parts of PBS 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, adding for a plurality of times while shaking for dissolving, and finishing adding within about 30 minutes); 3) then standing at 4 ℃ for two hours or overnight; 4) centrifuging at 12000rpm for 10min or 3000rpm for 20min, discarding the supernatant, and re-dissolving with PBS to the original volume; 5) repeating the steps 2 and 3; 6) centrifuging at 12000rpm for 10min or 3000rpm for 20min, discarding supernatant, dissolving with appropriate amount of PBS, determining concentration, detecting purity by SDS-PAGE, and adding 40-50% glycerol.

(5) Ascites titer determination

Ascites titer was measured by indirect ELISA method, and the cells were diluted 2-fold stepwise from 3 rd to 10 th wells with 0.01MpH7.2 PBS buffer. The 1 st hole uses the serum of the mouse diluted to 100 times during fusion as a positive control, the 2 nd hole uses the RPMI1640 complete culture solution as a 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 detection system is effective, when the OD value is more than or equal to 2 times the OD value of the negative control, the detection system is positive, otherwise, the detection system is negative. The dilution ratio corresponding to the positive hole with the lowest detection value is the ascites titer, and the ascites titer can reach 4096K (K, representing 1000,4096K representing 4096000).

(6) Antibody titer determination

The purified antibody is uniformly diluted to 1mg/ml by PBS buffer solution with 0.01MpH7.2, then diluted by 100 times to be used as the initial 1 st hole, and diluted by 2 times from the 2 nd hole to the 10 th hole. The serum of the mice is diluted to 100 times during fusion to be used as a positive control in the 11 th hole, the RPMI1640 complete culture solution is used as a negative control in the 12 th hole, 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 detection system is effective, and the antibody titer determination standard is as follows: and taking log (dilution) as an abscissa and an OD value as an ordinate to prepare a curve, wherein the curve equation is that y is min + (max-min)/(1+10^ ((logEC50-x) × Hillslope)), and fitting the curve by using sigmaplot software to obtain the titer is 10logEC 50. The results showed that the anti-APOE 4 point mutation monoclonal antibody titre was 4096K (K, indicated 1000,4096K for 4096000).

(7) The subtype identification of the monoclonal antibody is carried out by adopting a mouse monoclonal antibody immunoglobulin typing kit of Serotec company. And (4) properly diluting the purified monoclonal antibody, and detecting, wherein the operation is strictly carried out according to the kit instruction. The results of the tests are shown in Table 3 below (data source A: absorbance 450 nm):

TABLE 3 subtype test results

The monoclonal antibody secreted by the hybridoma cell line ACE8 is gamma 1 type, and has high specificity and sensitivity with the antigen peptide (amino acid sequence at positions 109-124) of APOE 4. The hybridoma cell strain ACE8 has been preserved by China Center for Type Culture Collection (CCTCC); address: wuhan university in Wuhan, China; the preservation date is as follows: 22 months 7 in 2020; the preservation number is: CCTCC NO: C2020121.

EXAMPLE 3 identification of monoclonal antibodies

(1) Immunoblot identification

pcDNA3.1(+) full-length expression plasmids containing nucleotide sequences of APOE4-112Cys, namely ApoE2(112Cys,158Cys) protein, ApoE3(112Cys,158Arg) protein and APOE4-112Arg, namely ApoE4(112Arg,158Arg) protein are respectively constructed, HEK-293 cells are respectively transfected by the plasmids, WesternBlotting detection is carried out on cultured cell lysate by using the monoclonal antibody, and a target band is presented at a corresponding position, which indicates that the expression of the ApoE4-112Arg protein is detected. The method comprises the following specific steps:

a. SDS polyacrylamide gel electrophoresis: see e.g.Oseber et al, eds molecular biology laboratory Manual (science Press 1998). The electrophoresis is terminated by adopting 10 percent separation gel and 5 percent concentrated gel under the electrophoresis condition of 150V voltage and the distance between the bromophenol blue dye band and the bottom edge of the gel is about 1.5 cm.

b. Electrotransfer: see e.g.Oseber et al, eds molecular biology laboratory Manual (science Press 1998). It was transferred to a PVDF (0.45 μm) membrane by electrotransfer.

c. Immunoblotting: after the electric membrane transfer is finished, the membrane is sealed in 5% skimmed milk powder sealing solution for 1 hour at room temperature, the monoclonal antibody (1 microgram/ml) prepared by the invention is used as a primary antibody, incubated for 2 hours at room temperature or reacted overnight at 4 ℃, and washed for 3 times with TBST (TBS added with 0.5% Tween-20) for 10 minutes each time. And (3) using horseradish peroxidase-labeled goat anti-mouse IgG as a secondary antibody, reacting at room temperature for 2h, washing by the method, acting on ECL for 1min, and exposing and imaging in a full-automatic chemiluminescence imager (Bio-Rad Versa doc5000 MP).

The results are shown in FIG. 2, and FIG. 2 shows Western blot analysis of extracts of transfected HeLa cells expressing full-length human ApoE2(hApoE2), ApoE3(hApoE3) or ApoE4(hApoE4), wherein the primary antibody used in the above figure is a monoclonal antibody produced by hybridoma ACE8 of the present invention, and the secondary antibody used in the following figure is a panApoE antibody (available from Eimei Biotech, Inc., Wuhan). ApoE2(112Cys,158Cys), ApoE3(112Cys,158Arg) were controls, none of which bound antibody, ApoE4(112Arg,158Arg) specifically bound antibody.

(2) Immunofluorescence cell staining identification

pcDNA3.1(+) full-length expression plasmids containing nucleotide sequences of APOE4-112Cys, namely ApoE2(112Cys,158Cys) protein, ApoE3(112Cys,158Arg) protein and APOE4-112Arg, namely ApoE4(112Arg,158Arg) protein are respectively constructed, the plasmids are respectively transfected into primary rat hippocampal nerve cells/human brain glioma U87 cells, and antibodies positive to APOE4-112Arg are verified according to an immunofluorescence technical process, and the results are shown in FIGS. 3 and 4.

(3) Immunohistochemical identification

Human skin, human kidney, human spleen and colon sections of some senile dementia patients (all obtained from the south-middle hospital of Wuhan university) are taken, and the sections are treated according to the following steps and then are placed under a microscope for observation:

tissue fixation: the slices were baked in a 60 ℃ incubator for 60 minutes prior to deparaffinization.

Dewaxing: the slices were soaked in xylene for 10 minutes, and then replaced with xylene and soaked for another 10 minutes.

Hydration: soaking in absolute ethyl alcohol for 5 minutes respectively; soaking in 95% ethanol for 5 min; soaking in 85% ethanol for 5 min; 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 solution (pH6.0) into pressure cooker, heating to boil, placing slices on heat-resistant plastic slice rack, placing into cooker, covering cooker lid, covering pressure valve, heating, stopping heating after the pressure valve bounces, starting timing, stopping heating after 1.5 min, opening cooker lid after pressure returns to zero, cooling solution to room temperature, and taking out slices.

Washing: ddH₂Soaking for 5min in O, washing for 2 times, soaking for 5min in PBST (500mL10 XPBS +4500mL ddH2O +5mL Tween-20), and washing for 2 times.

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

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

Adding a primary antibody: mu.l of diluted primary antibody (anti-APOE 4 point mutation monoclonal antibody, ACE 8-produced monoclonal antibody) was added and incubated in a wet box at 37 ℃ for 1 hour.

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

Adding Postblocking: add a 50. mu.l drop of reagent A to the histochemical kit (Ready to use histochemical kit from Mich.) and incubate in the wet box for 30 minutes at room temperature.

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

Adding an enzyme-labeled secondary antibody: a drop of 50. mu.l of reagent B (HRP-labeled secondary antibody) was added to the reaction mixture, and the mixture was incubated at 37 ℃ for 30 minutes.

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

Color development: DAB method: adding 0.85ml of distilled water into a DAB color development kit (purchased from Fuzhou Mixin Biotechnology Co., Ltd.) reagent A50 μ l, and mixing; adding 50 μ l of DAB color reagent kit reagent B, and mixing; adding a DAB color development kit reagent C50 mu l, and uniformly mixing; and (4) avoiding light. Drop wise onto the sections, 100. mu.l each wet box, and develop for 5 minutes.

And (3) stopping color development: the color reaction was stopped with distilled water.

Counterdyeing: placing the section into hematoxylin staining solution, staining for 30min, and washing with distilled water.

Sealing: DAB method: adding into hydrochloric acid methanol solution, and immediately washing with distilled water. Soaking in 75% ethanol for 5 min; soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min; soaking in anhydrous ethanol for 5 min. Soaking in xylene for 10min, replacing xylene, and soaking for 10 min. After air drying, the slices were covered with a cover slip.

And (4) observation: as a result of microscopic observation, FIGS. 5, 6, 7 and 8 were obtained, wherein in FIG. 5, A was a control peptide whose amino acid sequence was VERDVGRLRVQQGYEA, and B was an antigen-specific peptide whose amino acid sequence was EDVRGRLVQYRGEVQA, and both of the control peptide and the antigen-specific peptide were synthesized by a solid phase method.

(4) Determination of hybridoma cell strain ACE8 and APOE4 point mutation monoclonal antibody sequence

Extracting total RNA of a hybridoma cell strain ACE8, carrying out reverse transcription by using a universal primer to obtain cDNA, amplifying light chains and heavy chains of the antibody, separating the light chains and the heavy chains, cloning the light chains and the heavy chains to a standard cloning vector for expression, identifying the light chains and the heavy chains by using single colony PCR, selecting 5 single colonies with correct lengths of the light chains and the heavy chains for sequencing, and determining the single colonies as a real sequence of the antibody if the 5 sequencing results are almost the same. (sequencing and antibody Mass expression completion of Nanjing Kingsrei Biotech Co.)

The gene sequence result obtained by hybridoma cell strain ACE8 is as follows: the length of the heavy chain coding gene sequence of the APOE4 point mutation resisting monoclonal antibody is 1332bp, and the sequence is shown as SEQ ID NO. 1; the light chain coding gene sequence of the APOE4 point mutation resisting monoclonal antibody is 657bp long and is shown in SEQ ID NO. 2. Deducing a heavy chain coded by the gene sequence according to the obtained gene sequence, wherein the heavy chain consists of 444 amino acids, and the sequence is shown as SEQ ID NO. 3; the light chain consists of 219 amino acids, and the sequence is shown in SEQ ID NO. 4.

Indirect ELISA detection procedure:

1) taking a 96-well enzyme label plate, adding 50 mu l of immune complex with coating concentration diluted by coating solution into each well, namely 100ng of antigen, avoiding using edge holes as much as possible, reducing light absorption value to influence results, and incubating for 2 hours at 4 ℃ overnight or 37 ℃;

2) pouring out the antigen, adding 100 μ l of blocking solution (1% BSA, 0.1M KPi, 0.1% Tween-20, 0.02% thimerosal, pH7) into each well, incubating at 4 deg.C overnight or 37 deg.C for 2 hr, pouring out the blocking solution, washing the plate with washing solution (0.1M KPi, 0.05% Tween-20, pH7) for 3 times, and patting dry;

3) add 50 μ l of primary antibody per well, if hybridoma cell, then cell supernatant, if immune animal serum, then need to carry on a series of dilutions (blocking solution dilution), usually the initial dilution is 1:499, then 2 times after the dilution in turn, set up negative control, blank control and positive control each 2 wells, 37 degrees C incubation for 1 hour; washing the plate for 3 times, and patting to dry;

4) add 50. mu.l of 1: 1999 dilution (blocking dilution) of HRP-labeled secondary antibody, incubation at 37 ℃ for 45 min; washing the plate for 3 times, and patting to dry;

5) 100 μ l of TMB substrate (which must be ready-made) was added to each well and incubated for 5-20 minutes at room temperature.

6) Mu.l of stop buffer (0.5M oxalic acid) was added to each well and the absorbance at 450nm was read on a microplate reader.

The immunofluorescence technique flow comprises the following steps:

1. before operation, after the superclean bench irradiates for 20 minutes by ultraviolet rays, the fan is started for 5 minutes, and then subsequent operation can be carried out;

2. digesting primary rat hippocampal nerve cells/human brain glioma U87 cells (all purchased from Annuan (Wuhan) Biotech Co., Ltd.) with cell digestive juice, gently beating to prepare single cell suspension with cell concentration of 4 × 10⁵Inoculating a sterile 96-well cell culture plate at 100 mu l/well per ml;

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

4. Placing at 37 ℃ CO₂Culturing an incubator; cells were observed day by day and the results were recorded for 5-7 days.

5. Discarding liquid in a 96-well plate of primary rat hippocampal nerve cells/human brain glioma U87 cells transfected with plasmids (sucking negative holes first and sucking positive holes later), washing the plate for 2 times with PBS (PH 7.4), wherein each hole is 150 mu l, and beating the 96-well plate on absorbent paper after the plate washing is finished so that no residual liquid exists in the holes.

6. After the plate washing, 80% cold acetone was added to a 96-well plate in an amount of 150. mu.l per well, and the plate was fixed at-20 ℃ for 12 minutes.

7. After completion of the fixation, the fixative in the 96-well plate was aspirated off, and the plate was washed once with 150. mu.l/well of PBS at pH 7.4. After the plate washing is finished, the 96-hole plate is put on absorbent paper to be flapped, so that no residual liquid exists in the holes.

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

9. Add 3% BSA to 96-well plates, 150. mu.l per well, incubate at 37 ℃ for 30min, gently patte the liquid in the wells dry.

10. APOE4 point mutation monoclonal antibody was diluted with 1% BSA at a dilution ratio of 1: 1000 times.

11. Adding APOE4 point mutation monoclonal antibody into 96-well plate, placing 50 μ l of the monoclonal antibody into each well, and placing in 37 deg.C incubator for 1 hr

12. The plates were washed 3 times with PBST, 150ml per well, 3-5 min intervals.

13. And (3) diluting the FITC-labeled goat anti-mouse IgG secondary antibody at a dilution ratio of 1: 100 times (the second antibody should be protected from light)

14. The diluted secondary antibody was added to a 96-well plate at 50. mu.l per well and left at 37 ℃ for 1 hour.

15. The plate was washed 4 times with PBST, 150. mu.l per well, 3-5 min intervals, and finally 1 time with pure water, 150. mu.l per well, after washing, the 96-well plate was blotted dry on absorbent paper.

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

17. And (6) observing the result.

The reagents used were as follows:

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

Acetone, glycerol, Tween-80, KH2PO4, NaCl, KCl, Na2HPO 4.12H 2O, all of which are domestic analytical purities.

Preparation of washing 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 evenly and stored in a 200mL reagent bottle at the temperature of minus 20 ℃.

Example four

Double-sandwich ELISA kit

An ELISA kit for testing ApoE4 comprising:

the kit comprises a solid phase carrier containing a capture antibody, a sample diluent, a standard protein, enzyme-labeled primary anti-panApoE, a chromogenic substrate and a stop solution; the capture antibody was the antibody prepared in example two.

The capture antibody coating process was as follows:

(1) adding 100 μ L of 0.05M carbonic acid buffer solution with pH of 9.6 into 96-well enzyme label plate, diluting to obtain 10 μ g/L antibody solution, incubating at 4 deg.C overnight or 37 deg.C for 2 hr, and reserving several blank wells;

(2) pouring out the antibody solution, adding 100 μ l of blocking solution (1% BSA, 0.1M KPi, 0.1% Tween-20, 0.02% thimerosal, pH7) into each well, incubating at 4 deg.C overnight or 37 deg.C for 2 hours, pouring out the blocking solution, washing the plate with washing solution (0.1M KPi, 0.05% Tween-20, pH7) 3 times, and drying;

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

The standard protein is ApoE4 standard lyophilized powder (Projian biological (Wuhan) science and technology Co., Ltd.).

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

The stop solution is 2mol/L H₂SO₄And packaging into 5 ml/tube.

The enzyme-labeled primary antibody is a primary anti-panApoE labeled by horseradish peroxidase.

The preparation process comprises the following steps:

weighing horseradish peroxidase, dissolving in deionized water, and adding newly prepared 0.1M NaIO₄Stirring at room temperature in a dark place until the solution is brownish green;

dialyzed overnight at low temperature against 1M sodium acetate buffer pH 4.4.

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

adding 0.1ml of newly prepared 4mg/L NaBH₄The solution is mixed evenly and reacted for 2 hours at 4 ℃.

The above solution was filled into dialysis bag, dialyzed in 0.15M PBS pH7.4C, and overnight at 4 ℃.

10000g, centrifuging for 15min, discarding the precipitated protein, and obtaining the supernatant which is the enzyme-labeled anti-pan ApoE.

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

EXAMPLE five

Example four kit methods of use

1. Preparation of samples to be tested

Collecting human blood, placing in a centrifuge tube, centrifuging at 2500rpm for 10min after blood coagulation to obtain serum sample to be tested, storing at-20 deg.C to avoid repeated freeze thawing.

Preparation of Standard solutions

10 sample tubes were removed.

Standard solutions of different concentrations were prepared with sample dilutions and lyophilized powder for ApoE4 standard.

3. Test procedure

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

(2) incubating at 37 ℃ for 2 hours;

(3) after washing the plate for 4 times, patting the plate dry;

(4) adding 100 mu l of enzyme-labeled antibody or enzyme-labeled anti-pan ApoE solution diluted by 2000 times with the sample diluent, incubating for 2 hours at 37 ℃, washing the plate for 4 times, and then drying;

(5) adding 100 mul of TMB substrate color developing agent into each hole, and incubating for 15-20 min at room temperature. Add 100. mu.l of stop buffer per well and read the absorbance at 450nm on a microplate reader.

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

(7) and (4) substituting the degree of the sample to be tested into a curve, and calculating to obtain a test result.

EXAMPLE six

Test for detection accuracy

Standard weighing ApoE4 standard substance freeze-dried powder is added into sample diluent to prepare three groups of standard substance solutions with different concentrations, each standard substance solution is tested for three times according to the five testing processes of the embodiment to obtain the detection concentration, and the difference between the detection concentration and the nominal concentration is obtained by calculation, wherein the calculation formula is as follows: (detection concentration-nominal concentration)/nominal concentration) is 100%, and the detection results are 5.4%, 7.6% and 4.8%, which indicates that the kit has high test precision.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhan Tiande Biotechnology 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 (Homo sapiens)

<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(Homo sapiens)

<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(Homo sapiens)

<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(Homo sapiens)

<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(Homo sapiens)

<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(Homo sapiens)

<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(Homo sapiens)

<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 for testing ApoE4 protein content, wherein the ELISA kit for testing ApoE4 comprises:

the kit comprises a solid phase carrier containing a capture antibody, a sample diluent, a standard protein, the enzyme-labeled primary anti-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 shown as 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, respectively.

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

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

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

5. The ELISA kit of claim 1 for assaying the ApoE4 protein content, wherein the stop solution is 2mol/L H₂SO₄。

6. The ELISA kit for testing the content of ApoE4 protein according to claim 1 wherein the enzyme-labeled primary anti-panApoE is horseradish peroxidase-labeled murine monoclonal antibody panApoE.

7. The ELISA kit for testing the amount of ApoE4 protein according to claim 1 wherein the chromogenic agent is a TMB substrate chromogenic agent.