CN107058242A - The anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, monoclonal antibody and its preparation method and application, flow cytometer detection reagent - Google Patents
The anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, monoclonal antibody and its preparation method and application, flow cytometer detection reagent Download PDFInfo
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Abstract
The invention discloses a kind of anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, monoclonal antibody and its preparation method and application, flow cytometer detection reagent, belong to technical field of bioengineering.The present invention is using human peripheral leucocytes as immunogen immune Balb/c mouse, using cell-fusion techniques, utilize immunocytochemistry and streaming IIF screening hybridoma, the antibody produced using immunoprecipitation mass spectrography to monoclonal hybridoma carries out specificity identification, verified through Western Blot, the D5G8 of hybridoma cell strain II of one plant of energy anti-human CD61 monoclonal antibody of stably excreting mouse is obtained, deposit number is CGMCC No.13300.Monoclonal antibody is prepared using method is induced in animal body, it is IgG1 to identify heavy chain of antibody, light chain is kappa types, directly mark Immunofluorescence test human peripheral Thrombocyte CD61 antigen presentation available for streaming through FITC marks after antibody purification.
Description
Technical field
The present invention relates to a kind of anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, also relate to thin by the hybridoma
The anti-human CD61 monoclonal antibodies of mouse and the monoclonal antibody of FITC marks that born of the same parents' strain secretion is produced, and said monoclonal antibody
Preparation method and application, are additionally related to include the flow cytometer detection reagent of said monoclonal antibody, belong to biotechnology neck
Domain.
Background technology
CD61 alias CD61A, GPIIb/IIIa or Integrin β_3 chain, molecular weight is 110kDa, is expressed in blood platelet, macronucleus
On cell and macrophage.CD61 belongs to integrin beta subunit, its part and correlation molecule formula fibronectin, fiber egg
Bai Yuan, plasminogen, factor, TSP, glass connection albumen, vWF, tenascin, MMP-2, Collagen type Ⅳ and bone
Pontin protein.
The glass connection albumen that occurs when CD41/CD61 is platelet activation, the former albumen of fiber, fibrin, vWF by
Body, CD51/CD61 relevant with platelet aggregation be glass connection albumen, fibrinogen, fibrin, vWF, TSP by
Body is related to the adhesive reaction of cell.It can be clinically used for Glanzmann thrombasthenias, megakaryocytic leukemia, special hair
The diagnosis of property thrombocytopenic purpura.
Publication No. CN101200708A patent of invention discloses the anti-human CD41 monoclonal antibody hybridoma cells strain of mouse
CGMCC NO.2177, the anti-human CD41 monoclonal antibodies of mouse secreted by the hybridoma cell strain can recognize that CD41 and CD61 is tied
With GPIIb/IIIa compound co-immunoprecipitations under conformational antigen position after conjunction, non-reduced state, inhibiting antibody energy is used as
Enough suppress aggtegation caused by ADP, collagen and HIP2 etc., but do not influenceed on assembling caused by Ristocetin, this resists
Body can be used for the diagnosis and treatment for relating to blood platelet quasi-blood disease and thrombus disease.Publication No. CN101307109A patent of invention
The surface antigen monoclonal antibodies of mouse-anti-human T lymphocyte CD 3 are disclosed, by anti-T lymphocytes monoclonal antibody hybridoma cell
Strain CCTCC NO:C200803 secretes, and the antibody can be with known OKT3And WuT3Monoclonal antibody recognizes the different tables of antigen
Position, there is obvious activation, and performance dual regulation to the growth of normal bone marrow grain-macrophage progenitor cells (CFU-GM) system
Feature.At present, there is not the open source literature of the anti-human CD61 monoclonal antibody hybridoma cells strain of mouse also in the prior art.
The content of the invention
It is an object of the invention to provide a kind of anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, by the hybridoma
The anti-human CD61 monoclonal antibodies of mouse of strain secretion directly mark Immunofluorescence test human peripheral blood after being marked through FITC available for streaming
The expression of platelet CD61 antigens.
Secondly, the present invention provides a kind of anti-human CD61 monoclonal antibodies of mouse and its system secreted by above-mentioned hybridoma cell strain
Preparation Method.
Meanwhile, the present invention provides a kind of anti-human CD61 monoclonal antibodies of mouse of FITC marks and preparation method thereof.
Again, a kind of anti-human CD61 monoclonal antibodies of mouse of present invention offer, the monoclonal antibody of FITC marks are straight in streaming
Mark the application in Immunofluorescence test human peripheral Thrombocyte CD61 antigen presentation.
Finally, the present invention provides a kind of monoclonal comprising the anti-human CD61 monoclonal antibodies of above-mentioned mouse or FITC marks and resisted
The flow cytometer detection reagent of body.
In order to realize the above object the technical solution adopted in the present invention is：
The anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, entitled II D5G8, deposit number：CGMCC
No.13300, preservation date：On December 05th, 2016, depositary institution：China Committee for Culture Collection of Microorganisms is commonly micro-
Bio-Centers, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
The anti-human CD61 monoclonal antibodies of mouse, are secreted by the above-mentioned D5G8 of hybridoma cell strain II (CGMCC No.13300) and produced
It is raw.
The preparation method of the anti-human CD61 monoclonal antibodies of mouse, including：By the D5G8 (CGMCC of hybridoma cell strain II
No.13300 antibody is purified after) being seeded to mouse peritoneal, collection ascites, is produced.
The anti-human CD61 monoclonal antibodies of mouse directly mark Immunofluorescence test human peripheral Thrombocyte CD61 antigen presentation in streaming
In application.Specially：The above-mentioned anti-human CD61 monoclonal antibodies of mouse are marked using FITC, after being incubated with human peripheral blood platelet,
The expression of Immunofluorescence test human peripheral Thrombocyte CD61 antigen is directly marked using streaming.
The monoclonal antibody of FITC marks, marks the above-mentioned anti-human CD61 monoclonal antibodies of mouse to obtain using FITC.
The preparation method of the monoclonal antibody of FITC marks, comprises the following steps：Mark above-mentioned mouse anti-human using FITC
CD61 monoclonal antibodies, are produced.
The monoclonal antibody of FITC marks directly marks Immunofluorescence test human peripheral Thrombocyte CD61 antigen presentation in streaming
In application.Specially：After the monoclonal antibody that FITC is marked is incubated with human peripheral blood platelet, directly marked using streaming immune
The expression of fluoroscopic examination human peripheral Thrombocyte CD61 antigen.
Flow cytometer detection reagent, resists including at least the anti-human CD61 monoclonal antibodies of above-mentioned mouse or the FITC monoclonal marked
Body.
Beneficial effects of the present invention：
Present invention application human peripheral leucocytes are immunogene, Balb/c mouse are immunized, using classical cell fusion skill
Art, carries out positive-selecting, using immunoprecipitation-mass spectrography to Monoclonal hybridomas by immunocytochemistry to hybridoma
Cell carries out specificity identification, is verified through Western Blot, and obtaining one plant can the anti-human CD61 monoclonal antibody of stably excreting mouse
The D5G8 of hybridoma cell strain II, its deposit number：CGMCC No.13300, preservation date：On December 05th, 2016, preservation list
Position：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica of institute.
Method is induced in present invention use animal body and prepares monoclonal antibody, it is IgG1 to identify heavy chain of antibody, and light chain is kappa types.Adopt
Antibody is purified with Protein A affinity chromatographies, Immunofluorescence test people is directly marked available for streaming after being marked through FITC
The expression of peripheral blood Thrombocyte CD61 antigen.
Preservation is proved and survival proves explanation
Preservation cell line：The D5G8 of hybridoma cell strain II, deposit number：CGMCC No.13300, preservation date：2016
December 05, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1.
Brief description of the drawings
Fig. 1 is SDS-PAGE monoclonal antibody purity analysis results in embodiment 2；
Fig. 2 is the knot of FITC-CD61 monoclonal antibodies detection human peripheral Thrombocyte CD61 antigen presentation in embodiment 4
Really；
Fig. 3 is immunoprecipitation SDS-PAGE electrophoresis results in test example；
Fig. 4 is immunoprecipitation Western Blot result of the tests；
Fig. 5 is Abcam protein SDS-PAGE electrophoresis results；
Fig. 6 is that Western identifies IID5G8 antibody purification electrophoresis results；
Fig. 7 analyzes for the specificity of FITC-CD61 monoclonal antibodies.
Embodiment
Following embodiments are only described in further detail to the present invention, but do not constitute any limitation of the invention.
Embodiment 1
The D5G8 of hybridoma cell strain II preparation, comprises the following steps：
First, it is immunized
1st, the preparation of immunogene
(1) the fresh tunica albuginea that 3mL The Red Cross Blood Center, Henan Prov. fetches, plus 1 × PBS of 7mL dilutions is taken to mix；
(2) 15mL centrifuge tubes, plus 4mL lymphocyte separation mediums are taken, the tunica albuginea that 10mL dilutes slowly steadily is added on lymph
On cell separation liquid liquid level；
(3) horizontal 500 × g of refrigerated centrifuge, raising speed 1 are kept off, reduction of speed 0 is kept off, 18 DEG C, centrifugation 20min；
(4) ring-type milky-white layer is collected and with upper strata into new centrifuge tube；
(5) 1 × PBS to 14mL, 500 × g, 18 DEG C, centrifugation 20min are added, supernatant is abandoned；
(6) same to step (5) repeated washing 1 time；
(7) add appropriate 1 × PBS to be resuspended, 0.4% trypan blue solution is dyed, counted under microscope, adjust dense to required cell
Degree.
2nd, animal immune
Above-mentioned cell suspension, with 5 × 105Individual cell, 200 μ L, week old (6~8 week old of dorsal sc branch injecting immune 7
) female Balb/c mouse, immunization interval three weeks, altogether be immunized four times；First three day is merged with 5 × 107Individual cell (volume
200 μ L) tail vein injection carry out it is super exempt from, two exempt from, three exempt from, four exempt from after dock within three weeks and take the μ L of blood 10, be dissolved in 490 μ L 1 × PBS
In, 800 × g centrifugation 5min collect supernatant, -20 DEG C preserve in case antibody test.
2nd, titration
1st, streaming IIF detection antibody titer
(1) cell suspension is prepared：With the preparation of immunogene；
(2) antigen is added：With every pipe 1 × 106Individual cell adds to above-mentioned cell suspension in 5mL centrifuge tubes；
(3) primary antibody is added：The μ L/ of mouse immune serum 50 pipes of doubling dilution, if mice serum negative control pipe, PBS rather
Blank control pipe, 4 DEG C of incubation 60min；
(4) wash：Often pipe plus PBS 2mL, 500 × g centrifugation 10min, abandon supernatant, are vortexed and mix；
(5) secondary antibody is added：The sheep anti-mouse igg of BD companies PE marks, 5 μ L/ pipes, 4 DEG C of incubation 30min；
(6) wash：Often pipe plus PBS 2mL, 500 × g centrifugation 10min, abandon supernatant, plus 500 μ L PBS are resuspended；
(7) 300 mesh nylon net filters are into streaming loading pipe, upper machine testing.
2nd, testing result
2nd, three, four immunized mice serum carry out doubling dilution after diluting 100 times with 1 × PBS, and blood is not immunized while setting
Clear negative control and PBS blank controls, carry out streaming indirect immunofluorescene assay, to there is the maximum dilute of close percent positive
Multiple is released for antibody titer, is as a result shown：Two exempt from, three exempt from, four exempt from rear mice serum antibody titer and are followed successively by 1:800、1:1600、
1:3200。
3rd, cell fusion
1st, the culture of murine myeloma cell
The mouse myeloma cell line NS/0 cell lines frozen in liquid nitrogen are recovered in fusion the last fortnight, liquid is changed in good time,
Fusion expands culture in first 2 days, cell is reached cell number needed for fusion before fusion, and in exponential phase.Growth conditions
The perfectly round bright, size of good cell is homogeneous, and intracellular granular thing is few, without vacuole.
2nd, the preparation of feeder cells
(1) take Kunming mouse one to draw neck to put to death, be soaked in 75% alcohol 5 minutes；
(2) enter Cytology Lab, mouse is taken out, fixing limbs are on cystosepiment；
(3) suction pipe draw 30mL containing 1 × HAT (hypoxanthine (hypoxantin), aminopterin (aminopterin) and
Thymidine (thymidin)) 1640 complete mediums in plate；
(4) 20mL syringes are used to extract 1640 complete mediums (ibid) of the 12mL containing 1 × HAT from plate standby；
(5) mouse part skin is cut off, belly is fully exposed, lifts peritonaeum, 12mL is contained 1 × HAT's with syringe
1640 complete mediums are injected in pumpback 10mL after mouse peritoneal, soft mouse veutro wall, mix in plate, are prepared into 30mL
Feeder cells suspension；
(6) the feeder cells suspension prepared is sub-packed in 2 piece of 96 porocyte culture plates, 100 μ L/ holes；
(7) 96 orifice plates are deposited in into 37 DEG C, 5%CO2Cultivated in incubator.
3rd, cell fusion
(1) HAT culture mediums are taken out from 4 DEG C of refrigerators and GNK washing lotions is preheated into 37 DEG C of water-baths；
(2) super-clean bench is wiped, required experimental article, ultraviolet lower sterilizing super-clean bench and Cytology Lab is put into；
(3) oncocyte that four cell bottles grow fine is taken out, slowly holds up to make cell sink and blow and beat bottle wall for several times, used
Suction pipe shifts cell into 50mL centrifuge tubes, draws 500 μ L and counts, remaining 133 × g (1000r/min) centrifugations 10min；
(4) immune mouse is plucked into eyeball to put to death after sterilizing in 75% ethanol, blood is collected with 1.5mL EP pipes, in 37 DEG C
2h is placed, after after serum precipitation, 3000r/min centrifugation 5min draw supernatant and are used as positive serum；
(5) the mouse fixing limbs after alcohol-pickled move into super-clean bench, cut off with a set of sterile tweezer small on cystosepiment
Mouse epidermis, exposure peritonaeum, alcohol disinfecting, transducer set cuts off peritonaeum, takes out spleen and is put on 200 mesh nylon wires；
(6) GNK washing lotions are drawn with suction pipe and rinses nylon wire, prepare splenocyte suspension, open nylon wire by the spleen in beaker
Cell suspension moves to 50mL Li Xin ping, and GNK rinses beaker and collected into centrifugal bottle, adds GNK to 40mL, draws 500 μ L meters
Number, remaining 133 × g centrifugations 10min；
(7) splenocyte of centrifugation and NS/0 cells are abandoned into supernatant, added to GNK liquid in the centrifugal bottle containing splenocyte, blown and beaten
Mix, splenocyte is 1 with oncocyte mixed proportion:5；
(8) mixed 133 × g of cell is centrifuged into 10min, abandons supernatant；
(9) 37 DEG C of warm water of half cup are contained in plastic beaker on super-clean bench, knurl/spleen cell mixing centrifugal bottle is put into 37 DEG C of water
In bath, 1mL suction pipes draw 1mL PEG1500, are slowly uniformly added dropwise along centrifugation bottle wall while gently rotating centrifugal bottle, in 1min
Add；
(10) centrifugal bottle 1.5min is stood in water-bath；
(11) 15mL GNK liquid is added, rate of addition is 1mL 30s, 3mL 30s, 11mL 30s；
Centrifugal bottle 5min is incubated in (12) 37 DEG C of water-bath beakers, now cell mixing is located at bottom, GNK is located at cell suspension
On liquid level；
(13) GNK liquid 25mL, 133 × g centrifugation 10min is added into centrifugal bottle；
(14) supernatant is abandoned, flicks and breaks up cell mass, takes 30mL HAT1640 complete mediums into plate, is taken out from plate
Take 10mL culture mediums that cell mixing is resuspended, pumpback cell suspension is mixed into plate；
(15) the feeder cells plate prepared is taken, micro- Microscopic observation, growth conditions are fine；
(16) mixed cell suspension is added into the culture plate of 2 plate feeder cells plates and 1 plate without feeder cells with every μ L of hole 100
In, it is placed in 37 DEG C, cultivates in 5% CO2gas incubator.
4th, the selectivity culture of hybridoma
(hypoxanthine (hypoxantin), aminopterin (aminopterin) and chest containing 1 × HAT are used during cell fusion
Gland pyrimi piperidine deoxidating nucleus glycosides (thymidin)) selective medium, close observation cell growth state, the 4th day after cell fusion
There is less cloning cluster under microscope in visible part hole, background is more miscellaneous, liquid processing is changed in progress；After fusion the 7th day it is visible compared with
Obvious cloning cluster, background is gradually clear, is replaced by HT culture mediums；12nd day partial hole naked eyes visible white point-like gram after fusion
Grand group, is replaced by ordinary culture medium.
4th, the screening of hybridoma non-specificity and subclone
1st, the non-specific positive-selecting of hybridoma
Using streaming IIF, with reference to above-mentioned bioactivity, dilute serum primary antibody is replaced by hybridoma thin
Born of the same parents' culture supernatant, if super exempt from serum (1:1000) positive control, culture medium negative control, PBS blank controls, primary antibody sample-adding amount is
100 μ L/ holes, it is that testing result is positive fluorescent staining cell occur.
2nd, the monoclonal (limiting dilution assay) of hybridoma
(1) feeder cells are prepared；
(2) limiting dilution assay subclone hybridoma；
1. hybridoma is blown and beaten repeatedly, is counted after mixing, and adjustment cell number prepares 6.5mL cells and hanged in 20/mL
Liquid；
2. 100 μ L/ holes add to the row of A, B, C tri- of 96 hole feeder cells plates after mixing (i.e. per 2, hole cell)；
3. take 2.9mL cell suspensions to add 2.9mL complete culture solutions, cell number is 10/mL, 100 μ L/ holes add to D, E,
The rows of F tri- (i.e. per 1, hole cell)；
4. remaining 2.2mL cell suspensions are taken to add 2.2mL complete culture solutions, cell number is 5/mL, and 100 μ L/ holes add
To the row of G, H two (i.e. per 0.5, hole cell)；
5. 37 DEG C, 5%CO are put2Cultivated in incubator；
6. cultivate after 4 days (4~5 days) and observed on inverted microscope, it is seen that small cell clone, at the 10th day,
Naked eyes visible white point-like cell clone group, carries out culture supernatant antibody test；
7. the positive monoclonal cell strong, in good condition of selection detection turns 24 orifice plate cultures and repetition measurement, to positive hole by upper
State step and carry out 2 times, 3 subclones, until obtaining the basicly stable monoclonal cell strain IID5G8 of secretory antibody.
IID5G8 cell lines are frozen into conservation, sent in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, deposit number：CGMCC No.13300, preservation date：On December 05th, 2016, preservation address：The Chaoyang District, Beijing City North Star
No. 3 Institute of Microorganism, Academia Sinica of institute of West Road 1.
Embodiment 2
The preparation (mouse peritoneal induces method) of the anti-human CD61 monoclonal antibodies of mouse, comprises the following steps：
1st, prepared by ascites
(1) old female Balb/c mouse are chosen, atoleine injects mouse peritoneal after filtration sterilization, 500 μ L/ only,
It is standby to make；
(2) positive monoclonal cell line IID5G8 is transferred in Tissue Culture Dish and is enlarged culture, pneumoretroperitoneum injection in 7 days
Hybridoma, 0.8 × 106Individual/only, 4 mouse are inoculated with altogether, and injection dosage is 500 μ L/；
After (3) one weeks observe mouse ascites production, when the obvious projection of mouse web portion and tight skin, with 20mL without
Acicula head punctures mouse part skin, and ascites is gathered into centrifuge tube；
(4) ascites 3000r/min is centrifuged into 5min, suctions out central, clear liquid, packing, -80 DEG C of preservations.
2nd, AKTA protein chromatography systems antibody purification
(1) balance：(1 × PBS, pH are 7.4) to flow through albumin A affinity column to buffer solution；
(2) loading：The precipitation solution loading for taking 1mL ammonium sulfate two-step method to obtain；
(3) releveling：1 × PBS solution crosses albumin A affinity column；
(4) elute：Pillar is flowed through with the sodium citrate buffer solutions of 0.1mol/L pH 2.7 to be eluted；
(5) collect：Sample is connect with the Tris-HCl neutralization buffers of 1.0mol/L pH 9.0 and collects sample；
(6) dialyse：Purification sample is fitted into bag filter, and bag filter is placed in 1 × PBS into (pH is 7.4), pure through dialysis
Antibody volume is about 1.4mL after change；
(7) measure of concentration：BCA methods determine IID5G8 ascites, and concentration is 1.7mg/mL after purification；
(8) preserve：Sample storage is purified at -20 DEG C.
3rd, monoclonal antibody purity is verified using SDS-PAGE
Monoclonal antibody purity is analyzed using 15%SDS-PAGE combinations coomassie brilliant blue staining, as a result sees that M is in Fig. 1, figure
Marker, 1 be IID5G8 ascites before purification, 2 be IID5G8 ascites after purification.As shown in Figure 1, swimming lane 1 has more miscellaneous band, swimming lane
2 mainly have the heavy chain of antibody and the band of light chain two, illustrate that the purified rear purity of antibody is significantly improved.
Embodiment 3
The CD61 monoclonal antibodies of FITC marks, its preparation process is as follows：
(1) CD61 monoclonal antibodies (1mg/mL) to be crosslinked are taken to carry out AKTA purifying；
(2) FITC is dissolved in DMSO and (noted：Being crosslinked the FITC that uses every time all should Fresh, lucifuge), concentration is
1mg/mL；
(3) according to P:F (CD61 monoclonal antibodies:FITC) it is 1mg:FITC is slowly added in antibody-solutions by 150 μ g ratio, side
Edged is gently rocked, and it is well mixed with antibody, 4 DEG C of dark place reaction 8hr；
(4) 5mol/L NH is added4Cl to final concentration 50mmol/L, 4 DEG C of terminating reaction 2hr；
(5) cross-linking agent is dialysed more than four times in PBS, it is limpid to dialyzate；
(6) identification of cross-linking agent
Protein concentration (mg/mL)=(A280-0.31 × A495)/1.4；
F/P ratios：3.1 × A495/ (A280-0.31 × A495), the value should be between 2.5~6.5；
(7) albumen that FITC is crosslinked is placed in pH7.4 phosphate buffer, adds 0.1%NaN3, 1%BSA, 4 DEG C
Dark place is preserved.
NANODROP traces of albumin detecting instrument detects FITC-CD61 concentration：
(1) NANODROP trace of albumin detecting instruments are opened, ultra-pure water cleaning detection probe is added dropwise；
(2) 3 μ L albumen buffer solution (1 × PBS) are added dropwise in detection probe, clicks on blank and is calibrated；
(3) 3 μ L FITC-CD61 solution is added dropwise in detection probe, 488nm wavelength, reading numerical values is selected；
(4) FITC-CD61 is 0.04325mg/mL through NANODROP detectable concentrations.
Embodiment 4
The CD61 monoclonal antibodies of FITC marks are in Flow cytometry human peripheral Thrombocyte CD61 antigen presentation
Application, concrete operations are as follows：
1st, blood platelet is separated
(1) peripheral blood 1.5mL (EDTA anticoagulant blood-collectings pipe), is transferred in 2mL EP pipes；
(2) 200 × g centrifuges 20 minutes (minute, min) after mixing；
(3) take 2/3 plasma layer, in equal volume add PBS (pH value is 7.4), mix, 100 × g centrifugation 20min；
(4) supernatant is taken, 800 × g centrifugation 20min abandon supernatant；
(5) 800 × g of 2mL PBS centrifugations 20min is added to washed once, precipitation is blood platelet；
(6) hematoblastic concentration is detected.
2nd, streaming Immunofluorescence test Thrombocyte CD61 is expressed
(1) above-mentioned platelet suspension is taken, with every pipe 1 × 106Add in 2mL EP pipes, 100 μ L PBS works are added in No. 1 pipe
For negative control, the μ L of FITC-CD61 antibody 5 in embodiment 3 are added in No. 2 pipes, room temperature lucifuge is incubated 60min；
(2) each Guan Zhongjun adds upper machine testing after 300 μ L PBS are resuspended, and as a result sees that ((a), (b) are respectively No. 1 to Fig. 2 in figure
Pipe, No. 2 pipe testing results).
Figure it is seen that positive rate is 0.124% after the human peripheral blood platelet of separation is incubated with PBS, and embodiment
Positive rate is 97.0% after the 3 FITC-CD61 antibody prepared are incubated with human peripheral blood platelet.
Embodiment 5
Flow cytometer detection reagent, the anti-human CD61 monoclonal antibodies of mouse prepared comprising above-described embodiment 2 and other detection auxiliary agents,
It is prior art, here is omitted.
Embodiment 6
Flow cytometer detection reagent, the CD61 monoclonal antibodies of the FITC marks prepared comprising above-described embodiment 3 and other detections
Auxiliary agent.
Test example
First, the method for monoclonal antibody specificity identification
1st, test method
(1) NP40 cell pyrolysis liquid cell lysis is used, lymphocyte protein lysate is prepared, by 107Individual cell adds 1mL
NP40 cell pyrolysis liquids, are cracked after 1h on ice, and 4 DEG C, 10000 × g centrifugation 20min, supernatant are placed in -20 DEG C of preservations after packing；
(2) using lymphocyte protein lysate as protein sample, antibody is prepared with ascites, immunoprecipitation is carried out, exempts from
Epidemic disease precipitated product carry out SDS-PAGE electrophoresis (see Fig. 3, M is Marker in figure, 1 be protein G+ lymphocytolysises albumen+
IID5G8,2 be protein G, and 3 be lymphocytolysis albumen), coomassie brilliant blue R250 dyeing, while immunoprecipitation product
Carrying out Western Blot, (see Fig. 4, M is Marker in figure, and 1 is protein G+ lymphocytolysis albumen+IID5G8, and 2 are
Protein G, 3 be lymphocytolysis albumen), as a result carry out check analysis；
(3) immunoprecipitation product control western results, reclaim SDS-PAGE and examine dye glue purpose band, serve extra large new life
Life carries out mass spectral analysis, determines antigen title, and then determine antibody.
2nd, result of the test
(1) Western Blot and immunoprecipitation
From figure 3, it can be seen that 100kDa and 140kDa repeats position two clear bands in swimming lane 1.Can be with from Fig. 4
Find out, swimming lane 1 shows that antigenic site is 140kDa, and swimming lane 3 shows that antigenic site is 100kDa.Therefore by Fig. 3 swimming lanes 1
Band delivers to marine section's new life and determines mass spectrum at 100kDa and 140kDa positions.
(2) Mass Spectrometric Identification immunoprecipitating antigen
Qualification result shows that the corresponding 140kDa antigens mass spectrum of IID5G8 monoclonal antibodies is CD41, and 100kDa antigen mass spectrums are
CD61 (see the table below 1,2).
The corresponding 140kDa antigens Mass Spectrometer Method result of the IID5G8 monoclonal antibodies of table 1
The corresponding 100kDa antigens Mass Spectrometer Method result of the IID5G8 monoclonal antibodies of table 2
(3) monoclonal antibody specificity Western Blot are verified
It is CD61 and CD41 to identify that IID5G8 monoclonal antibodies immunoprecipitation obtains antigen through mass spectral analysis, therefore orders Abcam albumen
(containing natural CD41 and CD61) carries out Western identifications, and Abcam protein SDS-PAGE electrophoresis results are shown in that (M is Fig. 5 in figure
Marker, 1 is Abcam albumen), Western identifications IID5G8 antibody purification electrophoresis results are shown in that (a~d is followed successively by Fig. 6 in figure
IID5G8 antibody 1:200、1:500、1:1000、1:M is Marker in 2000 potency electrophoresis results, a~d, and 1 is that IID5G8 is pure
Change antibody).
From fig. 5, it can be seen that the Abcam albumen ordered removes ultrawhite containing CD41 and the hatching eggs of CD61 two, there are other miscellaneous eggs
In vain, but CD41 and CD61 row is bright of a relatively high.From fig. 6, it can be seen that IID5G8 antibody does primary antibody, in 95kD appearance on the upper side
Specific band, illustrates that IID5G8 antibody produces specific band for CD61 antigens.
2nd, monoclonal antibody subgroup identification
The anti-human CD61 monoclonal antibodies subclass of mouse is determined to capture ELISA method, is as a result shown：CD61 monoclonal antibody heavies
For IgG1, light chain is kappa types.
3rd, the monoclonal antibody CD61 of FITC marks specificity analysis
FITC-CD61 monoclonal antibodies in Example 3, immuno-fluorescence assay Thrombocyte CD61 table is directly marked using streaming
Reach, concrete operations are as follows：
1st, blood platelet is separated
(1) collection peripheral blood 1.5mL (EDTA anticoagulant blood-collectings pipe), is transferred in 2mL EP pipes；
(2) 800 revs/min (rotate per minute, r/min) is centrifuged 15 minutes after mixing；
(3) upper strata platelet rich plasma (Platelet-rich plasma, PRP) is taken, continues 2500r/min and centrifuges 5 points
Clock；
(4) supernatant discarding, adds BD 1 × hemolysin 1.5mL, after blood platelet is dispelled, and stands 10min destruction red blood cells；
(5) 2500r/min is centrifuged 5 minutes；
(6) supernatant discarding, plus 1 × PBS 2mL, and 2500r/min is centrifuged 5 minutes；
(7) operation of repeat step (6) is once；
(8) supernatant discarding, plus 1 × PBS resuspensions.
2nd, streaming Immunofluorescence test Thrombocyte CD61 is expressed
(1) above-mentioned platelet suspension is taken, with every pipe 1 × 106Add in 2mL EP pipes, 100 μ L PBS works are added in No. 9 pipes
Added for negative control, in No. 10 pipes in the μ L of FITC-CD61 antibody 10, No. 11 pipes and add CD61 ascites (1 after purification:1000 is dilute
Release, 100 μ L) be used as between mark positive control, room temperature lucifuge is incubated 60min；
(2) each Guan Zhongjun is added after 2mL PBS resuspensions, and 2500r/min is centrifuged 5 minutes；
(3) add to add in 5 μ L IgG-FITC, No. 10 pipes in 5 μ L PBS, No. 11 pipes in supernatant discarding, No. 9 pipes and add 5
μ L IgG-FITC, lucifuge is incubated 30min；
(4) each Guan Zhongjun is added after 2mL PBS resuspensions, and 2500r/min is centrifuged 5 minutes；
(5) each Guan Zhongjun adds upper machine testing after 300 μ L PBS are resuspended, and as a result sees that ((a) is No. 9 pipes to Fig. 7 in figure, and (b) is
No. 10 pipes, (c) is No. 11 pipes).
From figure 7 it can be seen that negative control is in negative range, FITC-CD61 antibody and the human peripheral blood of above-mentioned preparation
After platelet is incubated, positive rate is 95.7%, and through indirect fluorescent after unlabelled CD61 antibody and the incubation of human peripheral blood platelet
Dyeing, positive rate is 85.8%.Illustrate FITC-CD61 antibody FITC mark excellents prepared by embodiment 3, streaming can be used for
The expression of straight mark detection human peripheral Thrombocyte CD61 antigen.
Claims (10)
1. the anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, it is characterised in that：The hybridoma cell strain is II D5G8, is protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number：CGMCC No.13300, preservation day
Phase：On December 05th, 2016.
2. the anti-human CD61 monoclonal antibodies of mouse of hybridoma cell strain secretion as claimed in claim 1.
3. the preparation method of monoclonal antibody as claimed in claim 2, it is characterised in that comprise the following steps：Hybridoma is thin
Born of the same parents' strain purifies antibody after being seeded to mouse peritoneal, collection ascites, produces.
The monoclonal antibody of 4.FITC marks, it is characterised in that：Monoclonal antibody as claimed in claim 2 is marked to obtain using FITC
Arrive.
The preparation method of the monoclonal antibody of 5.FITC marks, it is characterised in that：It is as claimed in claim 2 single using FITC marks
Clonal antibody, is produced.
6. monoclonal antibody as claimed in claim 2 directly marks Immunofluorescence test human peripheral Thrombocyte CD61 antigen table in streaming
Application in reaching.
7. the monoclonal antibody of FITC marks as claimed in claim 4 directly marks Immunofluorescence test human peripheral blood platelet in streaming
Application in CD61 antigen presentations.
8. application according to claim 7, it is characterised in that：The monoclonal antibody that FITC is marked and human peripheral blood are small
After plate is incubated, the expression of Immunofluorescence test human peripheral Thrombocyte CD61 antigen is directly marked using streaming.
9. include the flow cytometer detection reagent of monoclonal antibody as claimed in claim 2.
10. include the flow cytometer detection reagent of the FITC as claimed in claim 4 monoclonal antibodies marked.
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| CN111454360A (en) * | 2020-04-14 | 2020-07-28 | 福州迈新生物技术开发有限公司 | anti-CD 61 protein monoclonal antibody, cell line, preparation method and application thereof |
| CN116178536A (en) * | 2022-11-22 | 2023-05-30 | 上海交通大学医学院附属瑞金医院 | Mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109517058A (en) * | 2018-10-24 | 2019-03-26 | 普健生物（武汉）科技有限公司 | A method of screening can apply to flow cytometry antibody |
| CN111454360A (en) * | 2020-04-14 | 2020-07-28 | 福州迈新生物技术开发有限公司 | anti-CD 61 protein monoclonal antibody, cell line, preparation method and application thereof |
| CN111454360B (en) * | 2020-04-14 | 2021-12-03 | 福州迈新生物技术开发有限公司 | anti-CD 61 protein monoclonal antibody, cell line, preparation method and application thereof |
| CN116178536A (en) * | 2022-11-22 | 2023-05-30 | 上海交通大学医学院附属瑞金医院 | Mouse anti-human IV type collagen alpha 5 chain NC1 segment monoclonal antibody and application thereof |
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