CN105132380B - The hybridoma that anti-NSE monoclonal antibodies generate - Google Patents
The hybridoma that anti-NSE monoclonal antibodies generate Download PDFInfo
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- CN105132380B CN105132380B CN201510454238.6A CN201510454238A CN105132380B CN 105132380 B CN105132380 B CN 105132380B CN 201510454238 A CN201510454238 A CN 201510454238A CN 105132380 B CN105132380 B CN 105132380B
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Abstract
The preparation for the hybridoma that anti-NSE monoclonal antibodies generate, belongs to field of immunoassay medicine.The present invention provides a kind of monoclonal antibody of specific recognition NSE, the hybridoma for generating the monoclonal antibody and the highly sensitive method using monoclonal antibody detection NSE.
Description
【Technical field】
The present invention relates to the hybridomas that NSE monoclonal antibodies generate.
【Background knowledge】
Neuronspecific enolase (neuron specific enolase, NSE) is by tri- subunit groups of α, β and γ
Into dimer, isodynamic enzyme can be divided into five kinds of α α, β β, γ γ, α β and α γ.α subunits are primarily present in the tissues such as liver, kidney；β
Subunit is primarily present in skeletal muscle and cardiac muscle；γ subunits are primarily present in nerve fiber.The isodynamic enzyme of γ γ, α γ composition is god
It is peculiar through member and neuroendocrine cell, therefore it is named as neuronspecific enolase (NSE) or γ-enzyme.NSE is a kind of acid
Property protease, isoelectric point be pH 4.7.NSE gene nucleotide series overall length 2423bp, open read frame is from the 226 to 1531st alkali
Base, length 1305bp encode 434 amino acid residues, molecular weight 78kDa.All antigenic determinants of NSE are all distributed in 48
~96,188~293,399~433 amino acid sequence areas positioned at its three-dimensional body structure surface, can stimulate body in immune response
Generate the monoclonal antibody of NSE, the detection available for NSE.NSE is specifically to be present in neuron, is enolase base
Because of one of superfamily member, glycolysis is primarily involved in, catalysis 2-phosphoglyceric acid becomes the key enzyme of phosphoenolpyruvate.
High concentration is present in the tumour cell that nerve cell and neuroendocrine cell and these cells are caused.Tumour cell sugar
Glycolytic activity increases extremely, and has the overexpression of NSE, especially in Small Cell Lung Cancer and neuroblastoma.NSE exists
Cell releases when being destroyed out of cytoplasm.Therefore the death toll of the NSE and neural endothelia derived cell in blood circulation
Amount is related, this is also the reason of NSE can be used as related neural endothelium source tumor markers.NSE is in Small Cell Lung Cancer clinic point
Phase also shows application value in terms of having good correlation, curative effect monitoring and judging prognosis.
【Invention content】
The purpose of the present invention is to provide a kind of anti-NSE monoclonal antibodies and establish a kind of simple and tool high sensitivity
The method for detecting NSE contents, this method can be applied to the detection of cancer.To solve the spirit of existing monoclonal antibody detection NSE
Sensitivity is inadequate or expensive, is not suitable for clinical large-scale the shortcomings that applying.
Present invention obtains the monoclonal antibody (mAb) that can generate specific recognition NSE hybridoma cell strain 9-1 with
Hybridoma cell strain 2-2, 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：C2015103 and CCTCC NO：
C2015104.In addition, by identification, two plants of monoclonal antibodies identify two different epitopes of NSE, pass through 9-1's and 2-2 respectively
It is a kind of highly sensitive and high-throughput detecting system with reference to double antibody sandwich enzyme immune response method is established.
Therefore, the present invention provides described below 1 to 3 content：
1. the hybridoma cell strain of anti-NSE, preserving number is respectively CCTCC NO：C2015103 and CCTCC NO：
C2015104。
2. the monoclonal antibody of anti-NSE is respectively CCTCC NO by preserving number：C2015103 and CCTCC NO：
Secreted by the hybridoma cell line of C2015104, the monoclonal antibody is named as 9-1 and 2-2.
3. the application of anti-NSE monoclonal antibodies as claimed in claim 2, that is, utilize the dual anti-of the monoclonal antibody
Body sandwich method ELISA detects NSE, and the antibody sources that sandwich method matches two-by-two are CCTCC NO in preserving number：C2015103 hybridizes
The antibody 9-1 and preserving number of knurl secretion are CCTCC NO：The antibody 2-2 of C2015104 hybridomas secretion, step include：
(1) it is coated with the monoclonal antibody 9-1 matched two-by-two；
(2) sample to be tested is added in be incubated；
(3) using another plant of monoclonal antibody 2-2 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-NSE monoclonal antibodies titre in ELISA method
Measurement result.
The anti-NSE 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
It measures and is mixed for the NSE of 30ug with Freund's complete adjuvant, PBS, completely after emulsification, 30ug every every time, takes back multiple spot and armpit
Under, groin is immunized.Immune programme：Second of same dose and incomplete Freund's adjuvant mixed immunity were carried out after 15 days；Again
Third time same dose and Freund's complete adjuvant mixed immunity are carried out after 15 days；Tail portion takes blood indirect ELISA method after 10 days
Serum titer is surveyed, 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 9-1 and 2-2.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 NSE.
2nd, the screening of monoclonal antibody
(1) by each cell of the NSE antigens of a concentration of 0.5ug/mL of 100uL to 96 orifice plates, make it 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 9-1 and 2-2, 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 9-1 and 2-2.
4th, the measure of antibody titer
The potency of 2 kinds of mAb filtered out is measured by ELISA method.9-1,2-2 (10ug/mL) are separately added into,
It after reaction, is developed the color using anti-mouse antibody and the TMB of horseradish peroxidase, two kinds of mAb potency reach 10-9With
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 NSE 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 NSE 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 NSE of initial concentration 1ug/mL
Antigen in incubation at room temperature 2 hours, then adds in another strain antibody (1: 5000 of HRP labels；100uL/ holes) and in incubation at room temperature
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-NSE matched, preserving number is respectively CCTCC NO：C2015103 and CCTCC NO：
C2015104。
2. the monoclonal antibody of anti-NSE matched, one plant of preserving number is CCTCC NO：The hybridoma cell line institute of C2015103
The monoclonal antibody of secretion is named as 9-1；Another plant of preserving number is CCTCC NO：Secreted by the hybridoma cell line of C2015104
Monoclonal antibody be named as 2-2.
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| CN102435748B (en) * | 2011-11-25 | 2014-07-16 | 广东药学院 | Double-antibody sandwich enzymelinked immunosorbent detection kit and preparation method thereof |
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