CN105132381A - Anti-HDL (high density lipoprotein) monoclonal antibody generation hybridoma - Google Patents
Anti-HDL (high density lipoprotein) monoclonal antibody generation hybridoma Download PDFInfo
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Abstract
The invention belongs to the medical field of immunoassay and relates to preparation of an anti-HDL monoclonal antibody generation hybridoma. The invention further provides a monoclonal antibody for specific recognition of HDL, the hybridoma for generation of the monoclonal antibody and a method of using the monoclonal antibody for detection of high sensitivity of HDL.
Description
[technical field]
The present invention relates to the hybridoma that HDL monoclonal antibody produces.
[background knowledge]
High-density lipoprotein (HDL) (HighDensityLipoprotein; HDL) be the plasma lipoprotein that blood Midst density is the highest, particle is minimum, its diameter is 7.5 ~ 10nm, and density is 1.21g/cm
3, containing 6% cholesterol, 13% cholesteryl ester and 50% protein, molecular weight is (1.5 ~ 3) × 10
6, its lipophorin is mostly aPoA.Synthesize in liver, intestines and blood, be responsible for endogenous cholesterol (based on cholesteryl ester) from tissue toward the antiport of liver.The height of plasma high density lipoprotein level content is negative correlation with the risk of trouble cardiovascular diseases.Because exportable cholesterol promotes the metabolism of cholesterol, so come into one's own as the kovakorisan factor now.Cholesterol in high-density lipoprotein (HDL) delivery surrounding tissue, then be converted into bile acide or directly discharged from enteron aisle by bile, angiography proves that high density lipoprotein cholesterol content and arterial lumen stenosis are significant negative correlation.So high-density lipoprotein (HDL) is a kind of antiatherogenic plasma lipoprotein, it is the protective factors of coronary heart disease.Be commonly called as " blood vessel street cleaner ".
High-density lipoprotein (HDL) is the basic substance of blood lipid metabolism, specially in vivo in conjunction with unnecessary blood fat, outside transporter, removes blood rubbish; And due to volume little, endarterium can be penetrated the cholesterol being deposited on the inside is disposed and carried out tube wall of dehematizing, repair the damaged cell of blood vessel endothelium, recover blood vessel elasticity, have and remove the hungry unnecessary blood fat of blood vessel, remove blood dirt, the effect of clean blood vessel.Normal blood lipid metabolism refers to that the quantity of high-density lipoprotein (HDL) and low-density lipoprotein or triglyceride level are proportional, namely high-density lipoprotein (HDL) molecule " can transport " 5-6 low-density lipoprotein or triglyceride level molecule carries out disaggregating treatment to liver, finally excretes.When the quantitative proportion of low-density lipoprotein or triglyceride level and high-density lipoprotein (HDL) is lacked of proper care, the indexs such as the total cholesterol in blood or triglyceride level will be made to exceed normal range, break former normal blood lipid metabolism balance, occur abnormal, the unnecessary blood fat that is called as " rubbish " will be bowed and be deposited on vessel wall, formed atherosis gradually, finally cause the generation of cardiovascular and cerebrovascular disease.Therefore say that the quantity of high-density lipoprotein (HDL) not only decides blood lipid metabolism in blood and whether balances, but also play and disappear or alleviate the fast vital role of atherosclerosis plaque.
[summary of the invention]
The object of the present invention is to provide a kind of anti-HDL monoclonal antibody and set up a kind of simple and method of tool highly sensitive detection HDL content, the method can be applicable to the detection of cancer.To solve the insufficient sensitivity or expensive that existing monoclonal antibody detects HDL, be not suitable for the shortcoming of clinical large-scale application.
Present invention obtains hybridoma cell strain 1-35-1 and the hybridoma cell strain 1-10-1 of the monoclonal antibody (mAb) that can produce specific recognition HDL, this 2 strain cell strain is preserved in China typical culture collection center (CCTCC) on July 17th, 2015 respectively, preservation address is, China. Wuhan. Wuhan University, deposit number is CCTCCNO:C201598 and CCTCCNO:C201599.In addition, through qualification, two strain monoclonal antibodies identify two different epi-positions of HDL respectively, establish DASELISA immune response method by the combination of 1-35-1 and 1-10-1, and it is a kind of highly sensitive and high-throughout detection system.
Therefore, the invention provides the content of described below 1 to 3:
1. the hybridoma cell strain of anti-HDL, its preserving number is respectively CCTCCNO:C201598 and CCTCCNO:C201599.
2. the monoclonal antibody of anti-HDL, respectively by preserving number secreted by the hybridoma cell line of CCTCCNO:C201598 and CCTCCNO:C201599, described monoclonal antibody called after 1-35-1 and 1-10-1.
3. the application of anti-HDL monoclonal antibody as claimed in claim 2, namely the DAS-ELISA of described monoclonal antibody is utilized to detect HDL, the antibody sources that sandwich assay matches between two in preserving number be CCTCCNO:C201598 hybridoma secretion antibody 1-35-1 and preserving number be CCTCCNO:C201599 hybridoma secretion antibody 1-10-1, its step comprises:
(1) with the described monoclonal antibody 1-35-1 bag quilt matched between two;
(2) add testing sample to hatch;
(3) another strain monoclonal antibody 1-10-1 marked using the described HRP matched between two resists as two, adds reaction system;
(4) add enzyme reaction substrate after washing, read OD value with 450nm;
(5) result shows that the sensitivity detected is very high.
[accompanying drawing explanation]
What accompanying drawing 1 showed is anti-HDL monoclonal antibody titre measuring result in ELISA method that the hybridoma in the present invention produces.
What accompanying drawing 2 showed is the anti-HDL monoclonal antibody titre measuring result marking HRP.
What accompanying drawing 3 showed is the detection sensitivity of sandwich method ELISA (S-ELISA) system.
[embodiment]
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition, should be understood that, after having set forth content of the present invention, those skilled in the art can do various change and amendment to the present invention, these equivalent form of values are the scope in the application defined in claims equally.
Embodiment 1: animal immune
Male Balb/C healthy mice about 8 week age of selection and myeloma cell's homology used, the HDL of antigen to be protein content be 30ug mixes with Freund's complete adjuvant, PBS, and after complete emulsification, 30ug is every only each, take back multiple spot, and oxter, inguinal region immunity.Immune programme for children: carried out second time same dose and Freund's incomplete adjuvant mixed immunity after 15 days; Third time same dose and Freund's complete adjuvant mixed immunity is carried out again after 15 days; After 10 days, afterbody is got blood indirect ELISA method and is surveyed serum titer, does not add adjuvant booster immunization with the pure antigen of same dose; After 3 days, extracting spleen cell merges.
Embodiment 2: the structure of hybridoma
1, the cultivation of myeloma cell strain and preparation
(1) what the present invention adopted is SP2/0 myeloma cell strain, and this cell strain growth and fusion efficiencies are all good, and the doubling time is 10-12 hour.Select during fusion to be in the good cell of logarithmic phase, cellular form and activity.Myeloma cell should first do to adapt to cultivate before fusion on substratum, makes Growth of Cells arrive best state (i.e. logarithmic phase);
(2) SP2/0 of cultivation is drawn in the pipe of 50mL, centrifugal, abandon supernatant, hang, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.
2, the preparation of splenocyte
(1) mouse is placed in sealing bag, fills CO
2treat its death by suffocation;
(2) mouse sterilization is fixed on dissection plate, in Bechtop, gets spleen, be placed in the culture dish of 12mL substratum, peel adhesion organization off, grinding spleen, till surplus white tissues, suction pipe all picks up, more slowly get make tissue block adhesion tube wall on, centrifugal, abandon supernatant, add the erythrocyte cracked liquid cracking 10min of 10mL, then the substratum adding 20-25mL stops its reaction, after centrifugal, abandon supernatant, add the substratum of 10mL, draw a small amount of 10 times of dilution countings.
3, cytogamy
Within after booster immunization 3 days, do cytogamy.
Cytogamy is the key link of hybridoma technology, and basic step gets the Sp2/0 cell that is in logarithmic phase and splenocyte 1: 10 mixes, by polyoxyethylene glycol (PEG) method to obtain hybridoma, and called after 1-35-1 and 1-10-1.The hybridoma obtained is suspended in the HAT substratum containing feeder cell, then joins in 96 orifice plates, at 37 DEG C, and 5%CO
2incubator in close cultivation 12 days.
Embodiment 3: the preparation of monoclonal antibody and screening
1, the preparation of monoclonal antibody
The Kong Gezhong of the hybridoma obtained from embodiment 2 reclaims the supernatant liquor of substratum, to be chosen in ELISA method the antigen reactive monoclonal antibody with HDL.
2, the screening of monoclonal antibody
(1) by 100uL concentration be each Kong Gezhong of HDL antigen to 96 orifice plates of 0.5ug/mL, after spending the night in 4 DEG C, make it be fixed on solid phase;
(2) closed 2 hours are carried out with the bovine serum albumin that 150uL concentration is 1%;
(3) medium supernatant of 100uL hybridoma is joined each Kong Gezhong, in 37 DEG C of reactions 2 hours, then add the sheep anti-mouse antibody of the horseradish peroxidase of dilution 10000 times in 37 DEG C of reactions 1 hour;
(4) tetramethyl benzidine microwell peroxidase substrate (TMB) is used to carry out colour developing 20min as substrate;
(5) adding 50uL concentration is after the sulfuric acid termination reaction of 0.1mol/L, measures the absorbancy of 450nm;
(6) select 1-35-1 and 1-10-1 that absorbancy is approximately 3, and carry out subclone by limiting dilution assay.
3, a large amount of preparation of monoclonal antibody and and purifying
Cell cell-culturing rotating bottle after subclone is carried out enlarged culturing, after about 20 days, collects supernatant, carry out affinitive layer purification with staphylococcal protein A,SPA (ProteinA).The monoclonal antibody obtained is called after 1-35-1 and 1-10-1 respectively.
4, the mensuration of antibody titer
Tiring of the 2 kinds of mAb filtered out is measured by ELISA method.Add 1-35-1,1-10-1 (10ug/mL) respectively, after the reaction, use the anti-mouse antibody of horseradish peroxidase and TMB to develop the color, two kinds of mAb tire and reach 10
-9above (shown in accompanying drawing 1).
Embodiment 4: the mark of monoclonal antibody and the mensuration of titre
To be purified into each antibody carries out HRP mark according to a conventional method, the titre of the monoclonal antibody marked is measured by method below, is that the HDL antigen of 0.5ug/mL is fixed on (100uL/ hole) on 96 hole microplates by concentration.Use the bovine serum albumin of 1% to carry out closed 2 hours, tagged monoclonal antibody (the first hole dilutes 100 times), from the second hole, do 4 times of dilutions, react 2 hours under room temperature.After adding TMB, reaction at room temperature carries out 20 minutes, by the sulfuric acid stopped reaction of 0.1mol/L.Measure the absorbancy at 450nm, obtain the titre for the antigen be fixed in solid phase by the mode in embodiment 3.Result shows to have effective titre (shown in accompanying drawing 2).
Embodiment 5: double-antibody sandwich elisa detects the foundation of HDL method
(1) with one of the described monoclonal antibody of matching between two bag quilt, the monoclonal antibody of 0.5ug/mL is added to microwell plate soil with the amount in 100uL/ hole, hatches be fixed on solid phase in 24 hours in 4 DEG C;
(2) pH value of lattice use in hole containing 0.1%Tween20 is the 20mMPBS (PBST) of 7.4, washs 2 times with the amount in 200uL/ hole.Add 1% bovine serum albumin with the amount in 150uL/ hole and carry out closed 2 hours;
(3) Kong Geyong PBST washs 4 times with the amount in 200uL/ hole, adds by the continuous 4 times of dilution HDL antigens of initial concentration 10ug/mL, in incubation at room temperature 2 hours, then adds another strain antibody (1: 5000 of HRP mark; 100uL/ hole) and in incubation at room temperature 2 hours;
(4), after adding TMB, reaction at room temperature carries out 20 minutes, and the sulfuric acid adding 0.1mol/L carrys out termination reaction and measures the absorbancy of 450nm;
(5) result shows the sensitivity of detection very high (shown in accompanying drawing 3).
Claims (3)
1. the hybridoma cell strain of anti-HDL, its preserving number is respectively CCTCCNO:C201598 and CCTCCNO:C201599.
2. the monoclonal antibody of anti-HDL, respectively by preserving number secreted by the hybridoma cell line of CCTCCNO:C201598 and CCTCCNO:C201599, described monoclonal antibody called after 1-35-1 and 1-10-1.
3. the application of anti-HDL monoclonal antibody as claimed in claim 2, namely the DAS-ELISA of described monoclonal antibody is utilized to detect HDL, the antibody sources that sandwich assay matches between two in preserving number be CCTCCNO:C201598 hybridoma secretion antibody 1-35-1 and preserving number be CCTCCNO:C201599 hybridoma secretion antibody 1-10-1, its step comprises:
(1) with the described monoclonal antibody 1-35-1 bag quilt matched between two;
(2) add testing sample to hatch;
(3) another strain monoclonal antibody 1-10-1 marked using the described HRP matched between two resists as two, adds reaction system;
(4) add enzyme reaction substrate after washing, read OD value with 450nm;
(5) result shows that the sensitivity detected is very high.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989009403A1 (en) * | 1988-03-29 | 1989-10-05 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
| US6193967B1 (en) * | 1992-10-02 | 2001-02-27 | Peter M. Morganelli | Bispecific reagents for redirected targeting of human lipoproteins |
| EP1142996B1 (en) * | 1998-12-22 | 2009-02-25 | Daiichi Pure Chemicals Co., Ltd. | Monoclonal antibody against apolipoprotein a-i |
-
2015
- 2015-07-25 CN CN201510454240.3A patent/CN105132381B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989009403A1 (en) * | 1988-03-29 | 1989-10-05 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
| US6193967B1 (en) * | 1992-10-02 | 2001-02-27 | Peter M. Morganelli | Bispecific reagents for redirected targeting of human lipoproteins |
| EP1142996B1 (en) * | 1998-12-22 | 2009-02-25 | Daiichi Pure Chemicals Co., Ltd. | Monoclonal antibody against apolipoprotein a-i |
Non-Patent Citations (1)
| Title |
|---|
| 张娟等: "氧化高密度脂蛋白和氧化低密度脂蛋白单克隆抗体的制备", 《实用医院临床杂志》 * |
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Inventor after: Liu Yuncheng Inventor after: Zhao Hongmei Inventor after: Li Ximei Inventor after: Hou Jie Inventor after: Dou Weiwei Inventor after: Dong Xiaofang Inventor after: Duan Huimin Inventor after: Sun Runsheng Inventor after: Chang Yuanyuan Inventor before: Li Chunlei Inventor before: Liu Yuncheng Inventor before: Li Ximei Inventor before: Hou Jie Inventor before: Dou Weiwei Inventor before: Dong Xiaofang |