CN106589124B - CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell - Google Patents
CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell Download PDFInfo
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Abstract
A kind of CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell, and CD146 monoclonal antibody is prepared by the hybridoma cell strain that deposit number is CCTCC NO:C2016183.CD146 monoclonal antibody 4F10 described in the method for the present invention has good specificity, can accurately identify tumor vessel pericyte, and CD146 monoclonal antibody 4F10 can be used for the fluidic cell sorting and detection of colloid knurl source pericyte.
Description
Technical field
The present invention relates to glioblastoma microenvironment and Anti-angiogenic therapy strategies, and relate more specifically to CD146 Dan Ke
Grand antibody detects and separates the application in identification in glioma perivascular cell.
Background technique
Glioblastoma is the common primary tumor of central nervous system, and grade malignancy is high, and patient survival is short, multiple
Hair rate is high, treatment is difficult.Tumor neogenetic blood vessels abundant are the important feature feature of glioblastoma, and lead to tumor proliferation
The major reason of invasion.Pericyte is the important component of capillary and capilary, is wrapped by basement membrane of blood vessel, and with it is interior
Chrotoplast has direct contact, plays a significant role in terms of stablizing blood vessel mechanical structure, maintaining endothelial cell physiologic function.State
Inside and outside early-stage study discovery, glioma perivascular cell is largely enriched with and there are height atypias.Seminar's current research hair
Existing, the glioma perivascular cell of 80%-90% is from glioma stem cells transdifferentiation.But it is effectively directed to due to lacking at present
The method of tumor vessel pericyte sorting and enrichment, phenotypic characteristic and the clinicopathologia meaning of glioblastoma medium vessels pericyte
Justice not yet illustrates.Therefore, the technical method of specific recognition tumor vessel pericyte is researched and developed, will be further understanding glioblastoma
Struma tumor perivascular cell genesis mechanism, its clear function phenotype and formulation targeted therapy strategy provide important support.
CD146 is a kind of single-stranded film connectivity glycoprotein, belongs to immunoglobulin superfamily member, with many cell adhesions point
Son has homology.One of the mark molecule for showing that CD146 is normal tissue medium vessels pericyte has been reported, it can specificity knowledge
Perivascular cell in other glioblastoma；But know for the specificity of CD146 epitope on tumorigenic perivascular cell
Other antibody has not been reported.
Summary of the invention
For above-mentioned problems of the prior art, the purpose of the present invention is to provide CD146 monoclonal antibodies in glue
Application in the detection of matter tumor perivascular cell and separation identification, preparation, which is directed to the specific antibody of CD146 and evaluates the antibody, to exist
Effect in sorting enrichment colloid knurl source perivascular cell and its phenotypic evaluation.
In order to achieve the above-mentioned object of the invention, The technical solution adopted by the invention is as follows:
A kind of CD146 monoclonal antibody, it is characterised in that the hybridoma for being CCTCC NO:C2016183 by deposit number
Strain preparation.
The CD146 monoclonal antibody is 4F10.
The preparation method of above-mentioned CD146 monoclonal antibody comprising the steps of:
1) immunogene prepares；
2) immunogen immune Balb/c mouse；
3) by immune Balb/c mice serum titration；
4) preparation of spleen bone-marrow-derived lymphocyte and SP2/0 cell suspension；
5) preparation of feeder cells；
6) myeloma cell and the fusion of spleen bone-marrow-derived lymphocyte；
7) screening of specific hybrid oncocyte；
8) prepared by CD146 monoclonal antibody ascites；
9) identification of CD146 monoclonal antibody subclass；
10) CD146 monoclonal antibody is purified in ascites；
11) ELISA identifies the specificity of CD146 monoclonal antibody.
Further, step 1) includes: the CD146 albumen using eukaryotic expression as immunogene (Met1-Gly559), very
The CD146 albumen of nuclear expression is that the CD146 albumen of people's engineering cell expression (is stuck up, article No.: 10115- purchased from Beijing Divine Land justice
H08H).
Further, step 2) includes carrying out first immunisation to Balb/c mouse, being immunized for second, be immunized and add for the third time
Strong immune, wherein CD146 albumen is mixed with isometric Freund's complete adjuvant emulsification and does immunogene by first immunisation, is exempted from for the second time
Epidemic disease and immune respectively mix CD146 albumen with isometric incomplete Freund's adjuvant emulsification of third time do immunogene, reinforce exempting from
CD146 albumen is done immunogene by epidemic disease, and the amount of each immunogene, volume injected and approach are constant.
Further, feeder cells are prepared using non-immune female Balb/c mouse in step 5).
Further, in step 9), CD146 monoclonal antibody has Ig subclass.
Further, in step 11), CD146 monoclonal antibody has specificity.
Further, comprising being sieved from CD146 monoclonal antibody with immunofluorescence technique with glioblastoma tissue staining
The CD146 for tumorigenic perivascular cell is selected in conjunction with the step of optimal CD146 monoclonal antibody, immunofluorescence technique
The following steps are included:
1) frozen tissue sections are taken out from -20 DEG C of refrigerators, room temperature rewarming 5 minutes；
2) it is sliced with PBS aquation, 5 minutes × 3 times；
3) it is closed and is sliced with 10% lowlenthal serum, is incubated at room temperature 30 minutes；
4) primary antibody is incubated for: the primary antibody after dilution is added, wherein CD146 monoclonal antibody (1:100), and Desmin monoclonal is anti-
Body (Abcam ab32362,1:100), CD248 polyclonal antibody (Abcam ab67273,1:50), CD31 monoclonal antibody
(CST#3528,1:100), (Abcam ab28364,1:50), 4 DEG C of overnight incubations；
5) next day is rinsed with PBS and is sliced, and 5 minutes × 3 times；
6) secondary antibody is incubated for: the fluorescence secondary antibody after dilution is added is incubated at room temperature 1-2 hours；
7) it is rinsed and is sliced with PBS, 5 minutes × 3 times；
8) DAPI dye liquor room temperature is incubated, and 20 minutes；
9) it is rinsed and is sliced with PBS, 5 minutes × 5 times；
10) it with anti-fluorescence quenching mounting, is placed in wet box and is kept in dark place for 4 DEG C；
11) confocal laser scanning microscope, acquisition image.
Further, CD146 monoclonal antibody is 4F10.
Application of the CD146 monoclonal antibody in the fluidic cell sorting and detection of colloid knurl source pericyte.
Application of the CD146 monoclonal antibody in preparation treatment people's glioblastoma tumor medicine.
The invention has the benefit that
1, it based on colloid knurl source perivascular cell, designs and prepares the specific antibody for CD146.Success structure
Build the best monoclonal antibody combined for tumorigenic perivascular cell CD146.
2, the CD146 monoclonal antibody 4F10 that immunofluorescence results show that present invention preparation screening obtains being capable of specificity
Identify that glioma perivascular cell, glioma perivascular cell cell tight are centered around outside the vascular endothelial cell of the CD31 positive
Layer, meets the layer of structure feature of blood vessel.Compared with the commercial antibodies of targeting CD248, Desmin pericyte marker, this hair
The CD146 antibody 4F10 of bright preparation has good specificity, can accurately identify tumor vessel pericyte.
3, the CD146 monoclonal antibody 4F10 that present invention preparation screening obtains can be used for the streaming of colloid knurl source pericyte
Cell sorting and detection.PCR result further confirms that the CD146 positive cell height expression pericyte that airflow classification obtains marks
Object Desmin, CD248；Result of the present invention provides effective laboratory facilities for the function phenotype of further research perivascular cell.
Detailed description of the invention
Fig. 1 is the specificity that ELISA identifies CD146 monoclonal antibody；
The indirect Elisa of A:4F10 and immunogen protein His-CD146 is tested, and can preferably be combined as the result is shown；B:
4F10 is tested from the indirect Elisa of the CD146 albumen FC-CD146 of different labels, can preferably be combined as the result is shown；C:4F10
It is tested with the indirect Elisa of the CD105 albumen His-CD146105 of same label, as the result is shown no cross reaction.
Fig. 2 is CD146 monoclonal antibody and commercially available antibody immunofluorescence dyeing；Wherein, 2A:4F10 and commercialization CD31 exempts from
Epidemic disease fluorescent staining, the glioma pericyte of 4F10 label is located on the outside of the endothelial cell of CD31 label as the result is shown；2B: commercialization
CD248 and commercialization CD31 immunofluorescence dyeing, CD248 is unable to specific mark glioma pericyte as the result is shown；2C: commercialization
Desmin and commercialization CD31 immunofluorescence dyeing, Desmin is unable to specific mark glioma pericyte as the result is shown.
Fig. 3 is that CD146 monoclonal antibody sorts glioma primary cell and detection；Wherein 3A:4F10 airflow classification colloid
Tumor primary cell, cell, which has, as the result is shown obviously divides group, can be divided into positive and two groups negative；3B: the glioma of airflow classification is former
Q-PCR detection is carried out for cell positive and negative group's cell, as the result is shown the more negative group's pericyte correlating markings of 4F10 positive group
Object expression is higher.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, right below with reference to examples and drawings
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
The preservation information of hybridoma cell strain of the present invention: depositary institution is China typical culture collection center, ground
Location is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University, and the deposit date is on October 30th, 2016, deposit number was CCTCC NO:
C2016183, culture title and dated diagnostic characteristics: anti-human CD146 hybridoma cell strain 4-F10 (IgGI).
The preparation method of CD146 monoclonal antibody of the invention, comprising the steps of: made using the CD146 albumen of eukaryotic expression
For immunogene, hybridoma antibody cell strain is prepared by cell-fusion techniques and purifies corresponding CD146 monoclonal antibody, in order to avoid
Epidemic disease fluorescent technique is filtered out from CD146 monoclonal antibody for tumorigenic perivascular cell with glioblastoma tissue staining
CD146 combine optimal CD146 monoclonal antibody.
Embodiment 1: the preparation of CD146 monoclonal antibody
1) immunogene prepares
It is stuck up using purchased from Beijing Divine Land justice, the His-CD146 albumen of (article No.: 10115-H08H) people's engineering cell expression
(Met1-Gly559) immunogen immune female Balb/c mouse is done, spleen bone-marrow-derived lymphocyte is obtained and is tested for hybridoma fusion.
2) immunogen immune Balb/c mouse
CD146 albumen to be taken out from -80 DEG C of refrigerators, Lowry method (Folin- phenol reagent process) measures protein content after dissolution,
The 10mmol/L PBS (phosphate buffered saline solution) of pH 7.4 is diluted to 1.0mg/ml.Choose 56 week old, weight about 20g
Female Balb/c mouse.Antigen emulsification is emulsified using antigen emulsifier.When first immunisation, by CD146 albumen and in equal volume
Freund's complete adjuvant emulsification mixing, every female Balb/c mouse add intraperitoneal injection by the intradermal multiple spot of amount of 100 μ g immunogenes.The
It carries out respectively second within 14 days and the 28th day and third time is immune, adjuvant uses incomplete Freund's adjuvant, amount of antigen, volume injected instead
Constant with approach, indirect elisa method measures potency after third time is immune.Merge first 3 days progress booster immunizations, every female
Balb/c mouse peritoneal injects the 100 μ g immunogene CD146 albumen that adjuvant is not added, cell fusion after 3 days.
3) by immune Balb/c mice serum titration
10 days after third time is immune, blood examination is taken to survey serum antibody titer from female Balb/c mouse tail vein；Use 0.1M
CD146 albumen is diluted to best effort concentration 3-5 μ g/ml by NaCO3 (PH9.6) coating buffer, and every hole adds 100 μ l antigen liquids, in 4
DEG C overnight, board-washing machine-wash plate 5 times, finally reaction plate is upside down on blotting paper, drains cleaning solution in hole.Blood sampling and diluted blood
It is clear: it pinches rat-tail, cuts a notch in tail vein with scissors after 75% alcohol disinfecting, 20 μ l of blood, 2000rpm is taken to be centrifuged 30 minutes,
It takes 1 μ l of supernatant that 999 μ l antibody diluents are added to mix, and carries out doubling dilution, it, will be diluted tested from 1:100 to 1:3200
100 μ l are added in the every hole of serum, while serum 1:100 dilution before female Balb/c mouse immune being taken to do negative control, antibody dilution
Liquid does blank control.37 DEG C are incubated for 1 hour, wash 5 times repeatedly or more；Horseradish peroxidase goat anti-mouse igg is diluted to
1:6000, every hole add 100 μ l, and 37 DEG C are incubated for 1 hour, wash 5 times；Add TMB (3,3', 5,5'- tetramethyl benzidine) developing solution
100 holes μ l/, room temperature dark place 3-5 minutes, every hole add 50 μ l of terminate liquid to observe as a result, terminating displaing yellow after TMB colour developing, use is enzyme-linked
Immune detector records 450nm reading, and 2.1 times that each hole OD value is greater than negative control OD value are surveyed after returning to zero with blank control wells,
It is as positive.Serum titer reaches 1:12800, can be used for cell fusion.
4) preparation of spleen bone-marrow-derived lymphocyte and SP2/0 cell suspension
A female Balb/c mouse good with CD146 protein immunization is taken, is extractd at female Balb/c eyeball of mouse bloodletting
Extremely, eye blood is collected after centrifugation the positive control that serum makees ELISA, and spleen is taken out in sterile working, is put into and fills 10ml and not exclusively train
In the glass dish for supporting base, washing carefully peels off the connective tissue and adipose tissue of surrounding, changes a glass dish, spleen is pulled out, set
It in 200 mesh stainless (steel) wires, is ground with the inner core of syringe, is rinsed with full culture medium is often cannotd be used up, pass through splenocyte
Mesh enters in solution, and splenocyte is moved in 10ml glass centrifuge tube, 1500rpm horizontal centrifugal 10 minutes, removes supernatant.Together
Method is cannotd be used up full culture medium 10ml and is washed cell 1 time, the cell of precipitating is collected by centrifugation, by cell 10ml incomplete culture medium
It is resuspended and mixes, cell count is about 1 × 108A cell.
SP2/0 cell is removed from liquid nitrogen, is put into rapidly in 37 DEG C of water-baths, constantly rocks, until cell solution is complete
Dissolution, cell is transferred in 10ml centrifuge tube, 1500rpm horizontal centrifugal 10 minutes, abandons supernatant, and 10ml complete culture solution is resuspended
Precipitating, cell suspension is transferred in 50ml culture bottle, sets 37 DEG C, 5%CO2It is cultivated in incubator.After cell well-grown
It is screened cell one week with the Selective agar medium containing 8-AG；First 2 days of fusion, reaches 4 bottles for 1 bottle of cell, then is merging same day cell just
In logarithmic growth phase, just, cell size is uniform for vigor, round and bright, the fusion same day, with connector bend dropping tube by SP2/0 cell
It gently blows down, is collected in centrifuge tube from tube wall, is centrifuged, abandon supernatant, after precipitating cannots be used up full culture medium washing, 10ml is endless
Full culture medium is resuspended, cell count, about 5 × 107It is a.
5) preparation of feeder cells
A non-immune female Balb/c mouse is taken, eyeball sacrificed by exsanguination is plucked, 70% ethyl alcohol soaking disinfection 5 minutes is cut
Female Balb/c mouse skin is opened, lifts peritonaeum with tweezers, cuts an osculum with scissors, connector bend dropping tube draws the incomplete training of pre-cooling
It supports base and rinses abdominal cavity, washing lotion is drawn in 50ml centrifuge tube.Same method cannots be used up full culture medium and rinses abdominal cavity 3 times, collects washing lotion,
1000rpm horizontal centrifugal 10 minutes at room temperature, remove supernatant, and 10ml incomplete culture medium is resuspended cell and counts.
6) myeloma cell and the fusion of spleen bone-marrow-derived lymphocyte
The PEG 1450 (polyethylene glycol 1450) of U.S. sigma is placed in pre-temperature in 37 DEG C of incubators before fusion, draw 1 ×
107A myeloma cell's suspension and 1 × 108A spleen bone-marrow-derived lymphocyte suspension (cell number 1:10) is to a 50ml sterile centrifugation tube
In, 30ml RPMI-1640 (Roswell Park Memorial Institute 1640) culture medium is added, is mixed well,
1500rpm is centrifuged 10 minutes, is abandoned supernatant, is flicked tube bottom, make cell mass that dropper loosely is used in 37 DEG C of water-baths of centrifuge tube at paste
The 50%PEG1450 solution for drawing 0.8ml pre-temperature, is slowly added into cell at from tube bottom about 2cm along tube wall, the rotation of side edged
Centrifuge tube was added at 1 minute or so, was then allowed to stand 90 seconds, and the 1640 culture medium 30ml that 37 DEG C of pre-temperatures are added dropwise terminate fusion,
It is added within 3 minutes, fast after speed is first slow, movement is soft, and centrifuge tube is stood 5 minutes in 37 DEG C of incubators, takes out centrifugation
Pipe, 1500rpm are centrifuged 5 minutes, discard supernatant, and 10ml HAT culture medium are added, cell is resuspended, gently blow and beat, mix, will merge
Cell inoculation is to 96 porocyte culture plates for being covered with feeder cells, and by 100 holes μ l/, every piece of culture plate stays 6 holes to be inoculated with SP2/0
Cell sets 37 DEG C, 5%CO as the negative control of HAT selection2It is cultivated in incubator.
The growing state of cell can be observed under inverted microscope, and add 100 μ of HAT culture medium within the 4-5 days after fusion
L can use indirect elisa method to survey hybridoma potency in the 10-12 days, and the changes HT culture medium culture for 14-15 days.
7) screening of specific hybrid oncocyte
12-15 days after fusion, when cell grows to the 1/4-1/2 bottom hole of full culture hole, is detected and trained using indirect elisa method
Support supernatant, screening positive clone；With CD146 albumen coated elisa plate (0.5 hole μ g/), 4 DEG C overnight, washing buffer washing 5
It is secondary, 5 minutes every time, liquid is patted dry, 0.2% PBST of 50 μ l cells and supernatants and 50 μ l is added (containing 0.05% tween
The phosphate buffer of pH 7.4), positive control selects the immune serum of mouse, and negative control selects SP2/0 culture supernatant, blank pair
According to cleaning solution, 37 DEG C are incubated for 1 hour；Wash ELISA Plate: 100 μ l of every hole addition contain the 0.1% diluted HRP label of PBST
Goat anti-mouse ig antibody (1:6000), 37 DEG C are incubated for 30 minutes；Washing, pats dry liquid, adds the 100 μ l/ of TMB solution of Fresh
Hole, room temperature dark place are reacted 3-5 minutes, and the every 50 μ l of hole of terminate liquid is added to terminate reaction, and microplate reader examines 450nm absorbance value.
As a result it is CD146 albumen express using people's engineering cell as antigen, Balb/c mouse 5, co-melting conjunction 2 is successively immunized
Secondary, successively 5 plants of CD146 antibody of screening acquisition high-affinity are secreted after 3 time clonings and ELISA screening altogether
The hybridoma cell strain of CD146 monoclonal antibody, these hybridomas are frozen for several times, and subculture in vitro separately culture 3 months or more
Energy stably excreting CD146 monoclonal antibody, finally freezes in liquid nitrogen container.
8) prepared by CD146 monoclonal antibody ascites
After filtering out positive colony, i.e., positive hybridoma cell should be carried out by cloning training using limiting dilution assay immediately
It supports, prepares feeder cells, be resuspended with 10ml incomplete culture medium, collect positive colony cell and count, cannot be used up full culture medium
Positive colony cell is diluted to 100/20ml, one piece is taken in advance added with 96 porocyte culture plates of feeder cells, is added
Remaining positive colony cell is transferred to expand in 24 orifice plates and cultivate, collects cell liquid nitrogen cryopreservation, together by 200 μ l cell suspensions
When by culture plate in 37 DEG C, 5%CO2Incubator culture, microscopically observation cell growth status after the 3rd day, gradually by cell
Expand culture.The paraffin oil to sterilize by the mouse of 8 week old or through producing mouse peritoneal injection, by 10 after about 10 days8A hybridoma
It is anti-for purifying to obtain by the way of extracting or killing cell mouse ascites in 10-14 days to mouse peritoneal for cell infusion
Body.
9) identification of CD146 monoclonal antibody subclass
Using Sigma Co., USA Mouse Monoclonal Antibody Isotyping Reagents (ELISA/
Ouchterlony Double Diffusion, Stock No.ISO-2 LOT 114k4817), operation by specification carries out.
(1) coated elisa plate: CD146 albumen is diluted to best effort concentration 5ug/ml with coating buffer, every hole adds 100 μ l
Antigen liquid, every plant plus 12 holes overnight in 4 DEG C are washed 5 times, and sky is dry.
(2) close: every hole adds 350 μ l of confining liquid, 37 DEG C incubations 1-1.5 hour, wash 5 times, it is empty to do.
(3) the fresh Hybridoma Cell Culture supernatant of 100 μ l is added in every hole or dilution monoclonal antibody, room temperature (20 DEG C -25 DEG C) are quiet
It sets 1 hour, shakes and wash 3 minutes, totally 5 times.
(4) IgG1, IgG2a, IgG2b, IgG3, IgM, IgA are diluted with 1:2000 with antibody diluent.
(5) diluted antibody, every kind of antibody add 2 holes to 100 μ l of every hole addition, are stored at room temperature 30 minutes, shake and wash 3 points
Clock, totally 5 times.
(6) diluted 100 μ l (1:1000) of rabbit-anti sheep IgG antibody is added in every hole, is stored at room temperature 15 minutes, shakes and wash 3 minutes,
Totally 5 times.
(7) 100 μ l substrate developing solutions are added in every hole, and room temperature is protected from light 10-15 minutes.
(8) 50 μ l terminate liquids are added in every hole, and observation is as a result, determine Ig subclass.
10) antibody purification in ascites
The ascites of fresh acquisition, 3000rpm/ divide 15 minutes, removing cell component (or the solids formed in frozen storage process
Matter) etc.；The ascites that upper layer is limpid is taken, 7.2 veronal buffered saline (VBS of PH is added in equivalent；0.004mol/L barbital,
0.15mol/L NaCl, 0.8mmol/L Mg2+, 0.3mmol/L Ca2+) dilution；Then to add in every 10ml dilution ascites
150mg SiO 2 powder mixes, and suspension shook frequently in incubation at room temperature 30 minutes；3000g is centrifuged 20 minutes, and lipid etc. is logical
Method removing is crossed, clear ascites can be obtained, antibody purification uses the HiTrap rProtein A of GE healthcare company
HP column.
(1) buffer needed for configuring antibody purification:
Binding Buffer (A liquid): 100mmol/L sodium phosphate (sodium phosphate), 100mmol/L citric acid
Sodium (sodium citrate), pH 7.0.
Elution Buffer (B liquid): 100mmol/L sodium phosphate (sodium phosphate), 100mmol/L citric acid
Sodium (sodium citrate), pH 3.0.
Assemble Buffer (C liquid): 1mol/L tri- (methylol) aminomethane (Tris-HCl), pH9.0.
(2) odd contradictive hydroperitoneum and A liquid are mixed with the ratio of 1:10,0.45 μm of membrane filtration waits loading.
(3) HiTrap rProtein A HP column is selected to access AKTA Explorer, A, B liquid sufficiently balance respective pipeline.
(4) sample for preparing (2) balances pillar with A liquid after A pipe loading, loading, then is purified by flash column with B liquid, receives
Collection eluting peak to being added in the collecting pipe of a small amount of C liquid in advance.
(5) it adjusts and washes Deproteinated pH to 7.0-8.0, antibody is dispensed into freezen protective.
11) ELISA identifies the specificity of CD146 monoclonal antibody
The eukaryotic expression CD146-Fc albumen (band Fc label protein, article No.: 10115-H02H) that purchase Beijing Divine Land justice is stuck up
And CD105-his albumen (band his label protein, article No.: 50407-M08H), albumen is used into coating diluted extremely respectively
100 μ l are added in the every hole of 5 μ g/ml, ELISA battens, and 4 DEG C of coatings are overnight.Next day takes out plank and abandons antigen, board-washing.By CD146 Dan Ke
Grand antibody 4F10 makees 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000 dilution,
It is added in corresponding hole by 100 holes μ l/, separately makees blank and yin-yang control wells.37 DEG C are incubated for 1 hour, board-washing, addition secondary antibody, and 1:
3000HRP marks goat anti-mouse IgG, is added in corresponding hole by 100 holes μ l/, and 37 DEG C are incubated for 40 minutes.Liquid is abandoned, board-washing is clapped
It is dry, 100 hole μ l/ of tmb substrate liquid is added, is protected from light colour developing 10 minutes, 50 hole μ l/ of terminate liquid is added.
As a result as shown in Figure 1, wherein A indicates that coating CD146 immune protein, B indicate coating CD146-Fc recombinant protein, C
Indicate coating CD105-His recombinant protein.CD146 monoclonal antibody 4F10 can preferably identify CD146-Fc, and nonrecognition
With the CD105-his albumen of belt transect his label, there is preferable specificity.
Embodiment 2: Immunofluorescence test
Using immunofluorescence technique, detect and CD146 monoclonal antibody more of the invention and commercialization pericyte antibody
Expression characteristic in people's glioblastoma tumor tissue and the total distribution situation with vascular endothelial cell label CD31.
1) frozen tissue sections are taken out from -20 DEG C of refrigerators, room temperature rewarming 5 minutes；
2) it is sliced with PBS (Zhong Shan Golden Bridge, ZLI-9062) aquation, 5 minutes × 3 times；
3) it is closed and is sliced with 10% lowlenthal serum (doctor's moral, AR0009), is incubated at room temperature 30 minutes；
4) primary antibody is incubated for: the primary antibody after dilution is added, wherein CD146 monoclonal antibody 4F10 (1:100), Desmin Dan Ke
Grand antibody (Abcam ab32362,1:100), CD248 polyclonal antibody (Abcam ab67273,1:50), CD31 monoclonal are anti-
Body (CST#3528,1:100), (Abcam ab28364,1:50), 4 DEG C of overnight incubations；
5) next day is rinsed with PBS and is sliced, and 5 minutes × 3 times；
6) secondary antibody is incubated for: fluorescence secondary antibody (Thermofisher, Cat#:A-11034, Cat#:A- after dilution is added
21422) it, is incubated at room temperature 1-2 hours；
7) it is rinsed and is sliced with PBS, 5 minutes × 3 times；
8) DAPI (the green skies, C1005) dye liquor room temperature is incubated, and 20 minutes；
9) it is rinsed and is sliced with PBS, 5 minutes × 5 times；
10) it with anti-fluorescence quenching mounting (the green skies, P0126), is placed in wet box and is kept in dark place for 4 DEG C；
11) laser confocal microscope (Leica, TCS-SP5) observation, acquisition image.
As a result as shown in Fig. 2, wherein 2A indicates CD146 monoclonal antibody, CD31 monoclonal antibody (Abcam
ab28364)；2B indicates CD248 polyclonal antibody (Abcam ab67273), CD31 monoclonal antibody (CST#3528)；2C is indicated
Desmin monoclonal antibody (Abcam ab32362), CD31 monoclonal antibody (CST#3528)；The positioning of CD31 staining endothelial cells
On the inside of CD146 monoclonal antibody 4F10, and it is not high to be commercialized Desmin, CD248 antibody dyeing specificity.The result shows that this
The CD146 monoclonal antibody 4F10 of invention can specific recognition glioma perivascular cell, glioma perivascular cell is close
It is centered around the vascular endothelial cell outer layer of the CD31 positive, meets the layer of structure feature of blood vessel.With targeting CD248, Desmin week
The commercial antibodies of cell marker are compared, and CD146 antibody 4F10 of the invention has good specificity, can be accurately identified
Tumor vessel pericyte.
Embodiment 3: airflow classification and detection
Using fluidic cell sorting technology, the separating effect of CD146 monoclonal antibody of the invention is measured.From people's glioma
CD146 positive cell and negative cells are separated in tissue, and pericyte marker in two groups of cells is compared using qRT-PCR technology
The differential expression of CD248, Desmin.
A. cell RNA (ribonucleic acid) extraction and concentration mensuration
1) by the CD146 positive and negative cells PBS (Zhong Shan Golden Bridge, ZLI-9062) cleaning 3 times after sorting, 1ml is used
RNAiso (Takara, 108-95-2) cracking, is placed in 1.5ml EP pipe；
2) 0.2ml chloroform (river east chemical industry) is added in each sample, acutely oscillation 20 seconds, and room temperature stands 5 minutes；
3) 12000g, 4 DEG C are centrifuged 15 minutes；
4) it takes upper layer to move into new 1.5ml EP pipe in the sample being layered, is added 250 μ l isopropanols (river east chemical industry),
It is reversed up and down for several times to mix, ice bath stands 20-30 minutes；
5) 12000g, 4 DEG C are centrifuged 15 minutes；
6) abandon supernatant, add 75% ethyl alcohol of 1ml (river east chemical industry dehydrated alcohol: DEPC water=3:1), up and down reverse for several times with
Washing precipitate；
7) 12000g, 4 DEG C are centrifuged 5 minutes；
8) removal ethyl alcohol, opening 1.5ml EP pipe pipe lid, drying at room temperature 10 minutes；
9) 20-50 μ l DEPC water (the raw work in Shanghai, A1201H0001) dissolution RNA is added, is placed in and places on ice.
10) RNA concentration is measured.RNA concentration=OD260 × extension rate × 0.04 μ g/ μ l
B.RT-PCR reaction
It is carried out using Takara company qRT-PCR kit, operating procedure and condition are carried out referring to specification, PCR detection
It is carried out on CFX96 fluorescence quantitative PCR instrument.
Primer sequence:
CD146 F-AAGCAGGAGATCACGCTACC
R-GATTCGGGGCTAATGCCTCA
Desmin F-AAATCCGGCACCTCAAGGATGAGA
R-TTTCTCGGAAGTTGAGGGCAGAGT
CD248 F-GACCACCACTCATTTGCCTGGAA
R-AGTTGGGATAATGGGAAGCTGGGT
GAPDH F-GGAGCGAGATCCCTCCAAAAT
R-GGCTGTTGTCATACTTCTCATGG
As a result as shown in figure 3, wherein 3A indicates that airflow classification CD146 positive primary cell, 3B indicate CD146 positive cell
The expression of pericyte Research of predicting markers is higher.The result shows that: CD146 monoclonal antibody 4F10 of the invention can be used for colloid knurl source
The fluidic cell of pericyte sorts and detection.PCR result further confirms, the high table of CD146 positive cell that airflow classification obtains
Up to pericyte marker Desmin, CD248.The above results provide effectively for the function phenotype of further research perivascular cell
Laboratory facilities.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.
Sequence table
<110>the First Affiliated Hospital of Third Military Medical University of PLA
<120>CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell
<130>
<160> 8
<170> Patent In version 3.2
<210> 1
<211> 20
<212> DNA
<213>CD146 upstream primer
<400> 1
aagcaggaga tcacgctacc 20
<210> 2
<211> 20
<212> DNA
<213>CD146 downstream primer
<400> 2
gattcggggc taatgcctca 20
<210> 3
<211> 24
<212> DNA
<213>Desmin upstream primer
<400> 3
aaatccggca cctcaaggat gaga 24
<210> 4
<211> 24
<212> DNA
<213>Desmin downstream primer
<400> 4
tttctcggaa gttgagggca gagt 24
<210> 5
<211> 23
<212> DNA
<213>CD248 upstream primer
<400> 5
gaccaccact catttgcctg gaa 23
<210> 6
<211> 24
<212> DNA
<213>CD248 downstream primer
<400> 6
agttgggata atgggaagct gggt 24
<210> 7
<211> 21
<212> DNA
<213>GAPDH upstream primer
<400> 7
ggagcgagat ccctccaaaa t 21
<210> 8
<211> 23
<212> DNA
<213>GAPDH downstream primer
<400> 8
ggctgttgtc atacttctca tgg 23
Claims (2)
1. a kind of CD146 monoclonal antibody, it is characterised in that: the hybridoma for being CCTCC NO:C2016183 by deposit number
Strain preparation, the CD146 monoclonal antibody are 4F10.
2. CD146 monoclonal antibody described in claim 1 is in the fluidic cell sorting of preparation detection colloid knurl source pericyte
With the application of reagent used in detection.
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| CN108129567B (en) * | 2018-01-09 | 2019-02-26 | 南京任诺药业有限公司 | A kind of Humanized anti-human CD146 monoclonal antibody having efficient neutralization activity |
| CN108588033B (en) * | 2018-04-24 | 2021-09-24 | 富恩生物技术（成都）有限公司 | Hybridoma cell strain, CD31 monoclonal antibody, preparation method and application |
| CN108715836B (en) * | 2018-05-31 | 2020-07-17 | 中山大学孙逸仙纪念医院 | Separation and bionic culture method of pericytes in tumor tissue |
| CN111122848A (en) * | 2019-12-30 | 2020-05-08 | 浙江工业大学 | ELISA kit for screening HGFR (human chorionic gonadotropin receptor) targeted antibody and application |
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| CN101245101A (en) * | 2008-01-31 | 2008-08-20 | 中国科学院生物物理研究所 | Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146 |
| CN102936283A (en) * | 2008-01-31 | 2013-02-20 | 中国科学院生物物理研究所 | Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method |
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| CN101245101A (en) * | 2008-01-31 | 2008-08-20 | 中国科学院生物物理研究所 | Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146 |
| CN102936283A (en) * | 2008-01-31 | 2013-02-20 | 中国科学院生物物理研究所 | Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method |
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