CN105132378B - The hybridoma that anti-ApoE monoclonal antibodies generate - Google Patents
The hybridoma that anti-ApoE monoclonal antibodies generate Download PDFInfo
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- CN105132378B CN105132378B CN201510454055.4A CN201510454055A CN105132378B CN 105132378 B CN105132378 B CN 105132378B CN 201510454055 A CN201510454055 A CN 201510454055A CN 105132378 B CN105132378 B CN 105132378B
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Abstract
The preparation for the hybridoma that anti-ApoE monoclonal antibodies generate, belongs to field of immunoassay medicine.The present invention provides a kind of monoclonal antibody of specific recognition ApoE, the hybridoma for generating the monoclonal antibody and the highly sensitive method using monoclonal antibody detection ApoE.
Description
【Technical field】
The present invention relates to the hybridomas that ApoE monoclonal antibodies generate.
【Background knowledge】
Apo E (Apolipoprotein E, ApoE) is made of 299 amino acid, and molecular weight 34kDa is blood
Starch very low density lipoprotein (LDL), high-density lipoprotein (HDL), the component of the lipoprotein such as chylomicron (CM), can with it is low close
It spends lipoprotein receptor to combine, play a role in the metabolism of plasma cholesterol and triglyceride, there are three hypotypes for ApoE albumen tool：
ApoE2, ApoE3 and ApoE4, difference lies in the Arg of 112 Cys and 158.ApoE3 is a kind of more universal hypotype,
112 are Cys, and 158 are Arg.ApoE2 is Cys at 112 and 158, and ApoEA is then in 112 and 158
Arg, only there are one the variation of amino acid between every two kinds of hypotypes, great variety functionally but has occurred in so small variation,
Structure-activity relationship between this structure and function of ApoE is deeply probed by people.ApoE2 then has protection to senile dementia
Effect, and it is related with hyperlipoprotememia.ApoE3 plays a crucial role to the performance of body normal physiological function.ApoE4 and old age
Dull-witted, angiocardiopathy frequently-occurring correlation, and have to the recovery of various brain trauma functions and negatively affect external and animal
Experimental study shows that the ApoE polymorphisms in blood plasma are related to the coronary heart disease, hyperlipoprotememia, atherosclerosis of human body.
And the ApoE in brain is a kind of polyfunctional molecule, in amyloid beta deposition with removing, stablizing tubulin structure, intracellular letter
Number conduction etc. plays a significant role in cell processes, and polymorphism is related to senile dementia.Therefore, from its knot of Molecular level study
Structure-activity relationship between structure and function is of great significance to pathogenesis, diagnosis and the treatment of exploring relevant disease.
【Invention content】
The purpose of the present invention is to provide a kind of anti-ApoE monoclonal antibodies and establish a kind of simple and tool high sensitivity
Detection ApoE contents method, this method can be applied to the detection of cancer.To solve existing monoclonal antibody detection ApoE
Sensitivity it is inadequate or expensive, the shortcomings that being not suitable for clinical large-scale application.
Present invention obtains the hybridoma cell strain 23-1 for the monoclonal antibody (mAb) that can generate specific recognition ApoE
With hybridoma cell strain 8-1, this 2 plants of cell strains are preserved in China typical culture collection center on July 17th, 2015 respectively
(CCTCC), preservation address is Chinese Wuhan Wuhan Universitys, and deposit number is CCTCC NO：C201594 and CCTCC NO：
C201595.In addition, by identification, two plants of monoclonal antibodies identify two different epitopes of ApoE, pass through 23-1 and 8-1 respectively
Combination establish double antibody sandwich enzyme immune response method, be a kind of highly sensitive and high-throughput detecting system.
Therefore, the present invention provides described below 1 to 3 content：
1. the hybridoma cell strain of anti-ApoE, preserving number is respectively CCTCC NO：C201594 and CCTCC NO：
C201595。
2. the monoclonal antibody of anti-ApoE is respectively CCTCC NO by preserving number：C201594 and CCTCC NO：C201595
Hybridoma cell line secreted by, the monoclonal antibody is named as 23-1 and 8-1.
3. the application of anti-ApoE monoclonal antibodies as claimed in claim 2 utilizes the double of the monoclonal antibody
Antibody sandwich ELISA detects ApoE, and the antibody sources that sandwich method matches two-by-two are CCTCC NO in preserving number：C201594 is miscellaneous
It is CCTCC NO to hand over the antibody 23-1 of knurl secretion and preserving number：The antibody 8-1 of C201595 hybridomas secretion, step include：
(1) it is coated with the monoclonal antibody 23-1 matched two-by-two；
(2) sample to be tested is added in be incubated；
(3) using another plant of monoclonal antibody 8-1 of the HRP matched two-by-two the labels as secondary antibody, reactant is added in
System；
(4) enzyme reaction substrate is added in after washing, OD values are read with 450nm；
(5) the result shows that the sensitivity of detection is very high.
【Description of the drawings】
Attached drawing 1 be shown the present invention in hybridoma caused by anti-ApoE monoclonal antibodies dripped in ELISA method
Spend measurement result.
The anti-ApoE monoclonal antibodies titre measurement result of label HRP is shown in attached drawing 2.
The detection sensitivity of sandwich method ELISA (S-ELISA) system is shown in attached drawing 3.
【Specific embodiment】
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Furthermore, it is to be understood that after present disclosure is elaborated, those skilled in the art
It can make various changes and modification to the present invention, such equivalent forms are equally models as defined in the claims in the application
It encloses.
Embodiment 1：Animal immune
The male Balb/C healthy mices of selection 8 week old homologous with myeloma cell used or so, antigen is albumen
Measure and mixed for the ApoE of 30ug with Freund's complete adjuvant, PBS, completely after emulsification, 30ug every every time, take back multiple spot and
Oxter, groin are immunized.Immune programme：Second of same dose and incomplete Freund's adjuvant mixed immunity were carried out after 15 days；
Third time same dose and Freund's complete adjuvant mixed immunity are carried out after 15 days again；Tail portion takes blood indirect ELISA side after 10 days
Method surveys serum titer, and adjuvant booster immunization is not added with the pure antigen of same dose；Extracting spleen cell is merged after 3 days.
Embodiment 2：The structure of hybridoma
1st, the culture and preparation of myeloma cell strain
(1) for the present invention using SP2/0 myeloma cell strains, the cell strain growth and fusion efficiencies are good, during multiplication
Between be 10-12 hours.Selection is in exponential phase, cellular morphology and active good cell during fusion.Myeloma cell is being melted
It should first make to adapt to culture on culture medium before conjunction, make cell growth to best state (i.e. exponential phase)；
(2) SP2/0 of culture is drawn in the pipe of 50mL, centrifuges, abandon supernatant, hang, add the culture medium of 10mL, drawn few
10 times of dilutions of amount count.
2nd, the preparation of splenocyte
(1) mouse is placed in hermetic bag, fills CO2Treat its death by suffocation；
(2) mouse disinfection is fixed on dissection plate, takes spleen in superclean bench, be placed on the culture dish of 12mL culture mediums
In, it peels adhesion organization off, grinds spleen, until surplus white tissues, suction pipe all picks up, then slowly getting glues tissue block
On tube wall even, supernatant is abandoned in centrifugation, the erythrocyte cracked liquid of 10mL is added to crack 10min, then the culture medium of 20-25mL is added to terminate
It is reacted, and after centrifugation, abandons supernatant, adds the culture medium of 10mL, is drawn a small amount of 10 times of dilutions and is counted.
3rd, cell fusion
Do cell fusion within 3 days after booster immunization.
Cell fusion is the key link of hybridoma technology, and basic step is to take the Sp2/0 cells in exponential phase
It is mixed with splenocyte 1: 10, by polyethylene glycol (PEG) method to obtain hybridoma, is named as 23-1 and 8-1.It is obtained
Hybridoma is suspended in the HAT culture mediums containing feeder cells, is then added in 96 orifice plates, at 37 DEG C, 5%CO2's
Closing culture 12 days in incubator.
Embodiment 3：The preparation and screening of monoclonal antibody
1st, the preparation of monoclonal antibody
The supernatant of culture medium is recycled in the cell of hybridoma obtained in from embodiment 2, is chosen at ELISA side
In method with the antigen reactive monoclonal antibodies of ApoE.
2nd, the screening of monoclonal antibody
(1) by each cell of the ApoE antigens of a concentration of 0.5ug/mL of 100uL to 96 orifice plates, make after being stayed overnight in 4 DEG C
It is fixed on solid phase；
(2) closing 2 hours is carried out with the bovine serum albumin(BSA) of 150uL a concentration of 1%；
(3) medium supernatant of 100uL hybridomas is added in each cell, in 37 DEG C react 2 hours, so
The sheep anti-mouse antibody for adding in the horseradish peroxidase for diluting 10000 times afterwards reacts 1 hour in 37 DEG C；
(4) colour developing 20min is carried out using tetramethyl benzidine microwell peroxidase substrate (TMB) as substrate；
(5) after the sulfuric acid of a concentration of 0.1mol/L of addition 50uL terminates reaction, the absorbance of 450nm is measured；
(6) it selects absorbance and is about 3 23-1 and 8-1, and pass through limiting dilution assay and be subcloned.
3rd, a large amount of of monoclonal antibody prepare and and purify
Cell after subclone is enlarged culture with cell-culturing rotating bottle, after about 20 days, supernatant is collected, with grape ball
Bacterium A albumen (Protein A) carries out affinitive layer purification.Obtained monoclonal antibody is respectively designated as 23-1 and 8-1.
4th, the measure of antibody titer
The potency of 2 kinds of mAb filtered out is measured by ELISA method.23-1,8-1 (10ug/mL) are separately added into,
After the reaction, it is developed the color using the anti-mouse antibody of horseradish peroxidase with TMB, two kinds of mAb potency reach 10-9
Above (shown in attached drawing 1).
Embodiment 4：The label of monoclonal antibody and the measure of titre
To be purified into each antibody carries out HRP labels according to a conventional method, the titre of the monoclonal antibody marked is under
The method in face measures, and the ApoE antigens of a concentration of 0.5ug/mL are fixed on 96 hole microplates (100uL/ holes).Use 1% ox
Seralbumin carries out closing 2 hours, and the monoclonal antibody (the first hole dilutes 100 times) of marking does 4 times since the second hole
Dilution is reacted 2 hours at room temperature.After adding TMB, reaction carries out 20 minutes at room temperature, is stopped with the sulfuric acid of 0.1mol/L
Reaction.The absorbance in 450nm is measured, the titre for the antigen being fixed in solid phase is obtained in the way of in embodiment 3.Knot
Fruit shows there is effective titre (shown in attached drawing 2).
Embodiment 5：Double-antibody sandwich elisa detects the foundation of ApoE methods
(1) with one of the monoclonal antibody matched two-by-two coating, the monoclonal antibody of 0.5ug/mL is with 100uL/
The amount in hole is added to microwell plate soil, is incubated 24 hours in 4 DEG C and is fixed on solid phase；
(2) cell is using the 20mM PBS (PBST) that the pH value containing 0.1%Tween 20 is 7.4, with 200uL/ holes
Amount washing 2 times.1% bovine serum albumin(BSA) is added in the amount in 150uL/ holes and carries out closing 2 hours；
(3) cell is washed 4 times with PBST with the amount in 200uL/ holes, is added in by the continuous 4 times of dilutions of initial concentration 10ug/mL
ApoE antigens in incubation at room temperature 2 hours, then add in another strain antibody (1: 5000 of HRP labels；100uL/ holes) and in room temperature
It incubates 2 hours；
(4) after adding TMB, reaction carries out 20 minutes at room temperature, adds in the sulfuric acid of 0.1mol/L to terminate reaction and survey
Measure the absorbance of 450nm；
(5) the result shows that the sensitivity of detection is very high (shown in attached drawing 3).
Claims (2)
1. the hybridoma cell strain of anti-ApoE matched, preserving number is respectively CCTCC NO：C201594 and CCTCC NO：
C201595。
2. the monoclonal antibody of anti-ApoE matched, one plant of preserving number is CCTCC NO：The hybridoma cell line institute of C201594
The monoclonal antibody of secretion is named as 23-1；Another plant of preserving number is CCTCC NO：Secreted by the hybridoma cell line of C201595
Monoclonal antibody be named as 8-1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178408A (en) * | 2007-12-05 | 2008-05-14 | 杭州浙大生科生物技术有限公司 | Apolipoprotein E ELISA reagent box and method of producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178408A (en) * | 2007-12-05 | 2008-05-14 | 杭州浙大生科生物技术有限公司 | Apolipoprotein E ELISA reagent box and method of producing the same |
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| Title |
|---|
| 抗人载脂蛋白单克隆抗体制备及免疫学特性的研究;张东升等;《中国病理生理杂志》;19920629;第8卷(第2期);第229-232页，参考摘要、第230页左栏第2段及第231页左栏第2段 * |
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