CN104212770A - Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof - Google Patents
Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof. A hybridoma cell strain producing the monoclonal antibody is collected in China Center for Type Culture Collection, and the collection number thereof is CCTCC NO:201211. The anti-human FcepsilonRIalpha subunit monoclonal antibody prepared by the invention can be combined with a human IgE high-affinity receptor FcepsilonRIalpha, and can be used for detecting FcepsilonRIalpha expression on the surface of a cell; and the monoclonal antibody is of great significance for prevention and treatment of mast cell and basophilic granulocyte related diseases such as allergic dermatitis, dermatomyositis, psoriasis and the like, and has great clinical application value. The hybridoma cell strain provided by the invention is favorable in cell strain growth vigor, uniform in cell body size, muddy or transparent and vigorous in division, and has a proliferative potential of infinite division.
Description
Technical field
The invention belongs to bioengineering field, particularly a kind of anti-human Fc ε R I α subunit monoclonal antibody and application thereof, and the hybridoma cell strain of secreting described anti-human Fc ε R I α subunit monoclonal antibody.
Background technology
IgE (immunoglobulin E) acceptor is anaphylactoid important mediators.Mastocyte and basophilic granulocyte are that type Ⅰ allergy disease and some mastocyte are diseases related as the main effects cell of allergic dermatitis, dermatomyositis, psoriatic etc.Anaphylactogen is combined with its specific IgE after the cell surface IgE receptors bind such as the mixture that forms and mastocyte and basophilic granulocyte and is made IgE acceptor that micro-gathering occur, and triggering signal conduction, comprises that protein kinase activates, phosphatidylinositols is hydrolyzed and Ca
2+flow etc., cause that cell medium and cytokine are synthetic, expression of adhesion molecule increases and cell stretches, film fold forms and finally causes cell degranulation, discharge vaso-active substance and some enzyme and cause smooth muscle contraction, mucus secretion, Blood pressure drop and tissue injury.IgE acceptor has two kinds of high and low avidity, and Fc ε R I (IgE high-affinity receptor) is its high-affinity receptor, is mainly distributed in mastocyte and basophil, and also there is a small amount of expression on eosinophilic granulocyte surface.Alpha subunit is the combining site of Fc ε R I and IgE.Therefore, anti-Fc ε R I α subunit antibody is a kind of important antibody, and it is expressed with the relation of disease, explores through the mastocyte activation of Fc ε R I mediation or suppress the aspects such as mechanism, research IgE and antibody or antagonist, Fc ε R I and antibody or antagonist and have great effect research Fc ε R I.
Summary of the invention
The technical problem to be solved in the present invention is exactly, immune effect not good problem not strong for existing anti-human Fc ε R I α subunit monoclonal antibody specificity, a kind of high specificity is provided, the anti-human Fc ε of mass producible R I α subunit monoclonal antibody and application thereof, and a kind of hybridoma cell strain of secreting this anti-human Fc ε R I α subunit monoclonal antibody.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of hybridoma cell strain, and described cell strain is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C201211.
The present invention also provides the daughter cell of hybridoma cell strain as above.This hybridoma grows fine, and big or small homogeneous is muddy bright, bright, divides vigorously, has the Reproductive activity of unlimited division.
The present invention also provides the application of above-mentioned hybridoma cell strain in the anti-human Fc ε R I α subunit monoclonal antibody of preparation.The application of the described field routine that bases on practicality, preferably for utilizing described hybridoma cell strain secretion or preparing anti-human Fc ε R I α subunit monoclonal antibody.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of anti-human Fc ε R I α subunit monoclonal antibody, this monoclonal antibody is produced by above-mentioned hybridoma cell strain secretion.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of preparation method of anti-human Fc ε R I α subunit monoclonal antibody as mentioned above, and described preparation method comprises the following steps:
(1) vitro culture hybridoma cell strain of the present invention obtains nutrient solution;
(2), by step (1) gained medium centrifugal, collect supernatant purifying and obtain anti-human Fc ε R I α subunit monoclonal antibody.
Wherein the described vitro culture of step (1) is the Vitro Culture Techniques of this area routine, the temperature of described cultivation is preferably 37 ℃, the time of cultivating is preferably 24 hours, wherein the described centrifugal speed of step (2) is preferably 3000rpm, and the centrifugal time is preferably 5 minutes.Collect supernatant liquor, purification process obtains mouse anti human Fc ε R I α subunit monoclonal antibody.Wherein said purification technique is the purification technique of this area routine, preferably for utilizing commercially available monoclonal antibody purification kit to carry out purification process to gained supernatant liquor.
The preparation method who the invention also discloses a kind of people Fc ε R I α subunit monoclonal antibody, this preparation method comprises the following steps:
(1) employment Fc ε R1 α subunit recombinant protein immune mouse, the splenocyte and the myeloma cell that get immune mouse are merged, and the cell quantity integration percentage of splenocyte and medullary cell is that 5:1～10:1 filters out the hybridoma to responding property of people Fc ε R I α subunit;
(2) gained hybridoma is carried out after subculture in vitro separately cultivation and enlarged culturing, in hybridoma supernatant or the animal ascites from injection hybridoma, obtain anti-human Fc ε R I α subunit monoclonal antibody.
The ratio that wherein splenocyte of the described immune mouse of step (1) and medullary cell merge is the integration percentage of this area routine, and the integration percentage of described splenocyte and medullary cell is more preferably: 8:1～10:1, is preferably 10:1.Wherein said medullary cell is the medullary cell of this area routine, and medullary cell is preferably SP2/0 cell.
Wherein the described subculture in vitro separately cultivation of step (2) is the subculture in vitro separately culture technique of this area routine, and the time that described subculture in vitro separately is cultivated is preferably more than six months.The acquisition methods of wherein said anti-human Fc ε R I α subunit monoclonal antibody is the acquisition methods of this area routine, and described method preferably comprises: get BALB/c mouse about 8 week age, abdominal injection 0.3～0.5ml paraffin oil, pneumoretroperitoneum injection 1 * 10 in 2 weeks
6the hybridoma that individual step (1) obtains, injects and gets 2～3ml ascites after 7～10 days, and the centrifugal 3min of 3000rpm collects supernatant liquor, obtains the mouse anti human Fc ε R I α subunit monoclonal antibody of purifying.
The present invention prepares the anti-human Fc ε of gained R I α subunit monoclonal antibody and can combine with people Fc ε R I α subunit specifically, therefore this monoclonal antibody can be used for preparing the detection reagent of people Fc ε R I α subunit expression, the expression of people Fc ε R I α subunit in qualitative or detection by quantitative cell.
For solving the problems of the technologies described above, four of the technical scheme that the present invention takes is: a kind of detection method of cell people Fc ε R I α subunit expression, and this detection method comprises the following steps:
(1) anti-human Fc ε R I α subunit monoclonal antibody of the present invention is contacted with separated cell to be detected or the lysate of this cell;
(2) by immunofluorescence analysis method, flow cytometry method, immunoblotting assay method, elisa assay method, immunohistochemical analysis method or immunoprecipitation analysis method, judged whether positive reaction.
Wherein the described cell of step (1) is the conventional cell using in this area.Described cell is preferably eukaryotic cell or prokaryotic cell prokaryocyte, is more preferably mammalian cell, is preferably the cell that derives from people, mouse or monkey.
The immunofluorescence analysis method that wherein the described immunofluorescence analysis method of step (2) is this area routine, wherein said Tissue Culture Plate is the Tissue Culture Plate of this area routine, is preferably 96 porocyte culture plates.Wherein said centrifugal speed is preferably 1000rpm, and the centrifugal time is preferably 5min; The concentration of described cell is preferably 5 * 10
6individual/ml; Be inoculated in 96 porocyte culture plates, the inoculum size of every porocyte is preferably 100 μ l; In every hole, the add-on of anti-human Fc ε R I α subunit monoclonal antibody is preferably 100～150 μ g, the temperature that temperature is bathed is preferably 20～30 ℃, the time that temperature is bathed is preferably 8～12 hours, after washing, abandon supernatant, every hole adds fluorescently-labeled dynamics to carry out temperature bath, described antibody is preferably sheep anti-mouse igg (H+L) fluorescence antibody, the temperature bath time is preferably 30 minutes, the temperature that temperature is bathed is preferably room temperature, described room temperature typically refers to 20～30 ℃, fluorescence microscope result.
The flow cytometry method that wherein the described flow cytometry method of step (2) is this area routine, wherein said centrifugal speed is preferably 1000rpm, the centrifugal time is 5min; The concentration of described cell is preferably 2 * 10
5individual/ml; In cell, the add-on of anti-human Fc ε R I α subunit monoclonal antibody is preferably 100～150 μ g, the temperature that temperature is bathed is preferably 4 ℃, the time that temperature is bathed is 45 minutes, after washing, abandon supernatant, add fluorescently-labeled dynamics to carry out temperature and bathe, described antibody is preferably sheep anti-mouse igg (H+L)-PE fluorescence antibody, the temperature that temperature is bathed is preferably 4 ℃, the time that temperature is bathed is 45 minutes, abandons supernatant, upper machine testing after washing.
For solving the problems of the technologies described above, five of the technical scheme that the present invention takes is: the purposes of monoclonal antibody as above in preparing anti-allergy dermatitis, dermatomyositis or psoriasis.
Medicine of the present invention is a kind of composition, and described composition at least comprises a kind of activeconstituents: i.e. monoclonal antibody of the present invention and a kind of pharmaceutical carrier.In this pharmaceutical composition, monoclonal antibody of the present invention can be separately or together with other compounds as activeconstituents, wherein said " activeconstituents " refers to have prevention or treatment allergic dermatitis, dermatomyositis or psoriatic function.Described pharmaceutical carrier comprises pharmaceutically acceptable vehicle, weighting agent, thinner etc.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is: anti-human Fc ε R I alpha monoclonal antibodies prepared by the present invention can be combined with people IgE high-affinity receptor Fc ε R I α, can be applicable to detect the expression of cell surface people Fc ε R I α, this monoclonal antibody, for significant with mastocyte and the diseases related control as allergic dermatitis, dermatomyositis, psoriatic etc. of basophilic granulocyte, has high fundamental research value and clinical value.Hybridoma cell strain provided by the invention grows fine, and cell space size homogeneous is muddy bright bright, divides vigorously, has the Reproductive activity of unlimited division.
the preservation of biomaterial
Hybridoma cell strain provided by the invention has been deposited in Chinese Typical Representative culture collection center (CCTCC) on February 8th, 2012, preservation address: China. Wuhan. Wuhan University's postcode: 430072, this culture name is called hybridoma cell strain B4C2, and the deposit number of this hybridoma cell strain is CCTCCNO:C201211.
Accompanying drawing explanation
Fig. 1 is flow cytometry screening hybridoma cell strain positive colony figure.
Fig. 2 is that Western blot identifies anti-human Fc ε R I α subunit monoclonal antibody result figure.
Fig. 3 is that anti-human Fc ε R I α subunit monoclonal antibody detects mastocyte result figure in tonsil.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to ordinary method and condition, or selects according to catalogue." room temperature " described in embodiment refers to the temperature of the operation room of testing, and is generally 20～40 ℃.
Instrument and reagent source used in embodiment are as described below:
Flow cytometer is purchased from Beckman company, and fluorescent microscope is purchased from Olympus company;
People Fc ε R I α subunit recombinant protein is purchased from Hunan Yuan Tai company;
Freund's complete adjuvant and Freund's incomplete adjuvant are purchased from Sigma company;
DMEM, foetal calf serum, HAT substratum and HT substratum are purchased from Gibco company;
Goat anti-mouse igg antibody, DMSO, polyoxyethylene glycol and the bovine serum albumin of the sheep anti-mouse igg of FITC mark (H+L) fluorescence antibody, sheep anti-mouse igg (H+L)-PE fluorescence antibody, HRP mark are purchased from Sigma company;
Murine myeloma cell SP2/0 is purchased from cell institute of the Chinese Academy of Sciences;
DAB nitrite ion and phosphate buffered saline buffer are purchased from green skies company;
Hematorylin dye liquor is purchased from BASO company, and NAb Protein G Spin Kit is purchased from Pierce company.
The monoclonal antibody of the anti-human Fc ε of embodiment 1 R I α subunit and the preparation of secreting the hybridoma cell strain B4C2 of this antibody
1. animal immune
Employment Fc ε R I α subunit recombinant protein immunity body weight 18-20g Balb/c female mice in 6 week age (buying from Hunan Si Laike Jing Da laboratory animal company limited), get people Fc ε R I α subunit recombinant protein (concentration of this subunit recombinant protein is 0.1 μ g/ml) 0.5ml mix with equal-volume Freund's complete adjuvant fully emulsified after, through every of back of the body subcutaneous abdomen multi-point injection 0.5ml, 3 weeks, interval, get 0.5ml people Fc ε R I α subunit recombinant protein (concentration of this subunit recombinant protein is 0.1 μ g/ml) add equal-volume Freund's incomplete adjuvant mix fully emulsified after, abdominal injection 0.5ml is every for the second time.Immunity is for the third time carried out at interval after 2 weeks, the 4th immunity carried out at interval after 2 weeks, and the 5th immunity carried out at interval after 3 weeks, the 6th immunity carried out at interval after 3 weeks, method is all with for the second time, in cytogamy first 3 days, get people Fc ε R I α subunit recombinant protein 50 μ g abdominal injections, do last immunity.
2. cytogamy
Get the splenocyte of above-mentioned immune mouse and myeloma cell (SP2/0) in cell quantity than the ratio of 10:1, in the DMEM of serum-free (buying from Gibco) substratum, mix, the centrifugal 8min of 1500rpm, remove substratum, adding 0.5ml volume percent is that 50% PEG is (using PEG as fusogen, this reagent is bought from Sigma) make mixed solution, under 37 ℃ of water-baths, make it merge 2min, with the DMEM substratum of serum-free, stop merging the rear centrifugal 5min of 1000rpm, precipitation suspends with HAT substratum, divide and install in the cell plate that contain feeder cell in 96 holes, every hole addition is 200 μ l, 37 ℃, 5%CO
2cell culture incubator in cultivate.
3. the positive hole sizer of hybridoma selects and clone
In cell culture incubator, cultivate after 7 days, with HAT nutrient solution, carry out half amount and change liquid, after 2 weeks, with HT nutrient solution, change liquid, 3 Zhou Houke are changed to the DMEM nutrient solution with amount, when fused cell coverage hole floorage 25%～50%, adopt conventional ELISA method to screen positive hole, select the cell hole of strong positive reaction, carry out limiting dilution assay cloning cultivation, obtain the hybridoma cell strain B4C2 of the anti-human Fc ε R I α subunit monoclonal antibody of secretion.Through 6 months subculture in vitro separately and 30～50 times repeatedly after cryopreservation resuscitation cell strain all can well grow, and stably excreting antibody.Hybridoma cell strain is preserved for ascites preparation and liquid nitrogen after enlarged culturing.By gained hybridoma cell strain B4C2 be submitted to Chinese Typical Representative culture collection center carry out preservation (preservation address: China. Wuhan. Wuhan University's postcode: 430072), its deposit number is CCTCC NO:C201211.
4. monoclonal antibody ascites is prepared and purifying
Get BALB/c mouse about 8 week age, abdominal injection 0.5ml paraffin oil, pneumoretroperitoneum injection 1 * 10 in 2 weeks
6the B4C2 cell of the anti-human Fc ε of individual secretion R I α subunit monoclonal antibody (the positive hole sizer choosing of above-mentioned steps 3 hybridomas and clone thereof prepare gained), inject 7 days afterwards visible mouse web portion obviously expand, adopt mouse ascites, the centrifugal 3min of 3000rpm, collect supernatant liquor, be anti-human Fc ε R I α subunit monoclonal antibody (by its called after: MAb-B4C2), with-70 ℃ of preservations after NAb Protein G Spin Kit (buying from Pierce company) purifying.
Utilize ELISA method to measure tiring of gained antibody, this titer detection method is: by people Fc ε R1 α subunit antigen coated liquid (carbonate buffer solution 50mmol/L for recombinant protein, pH 9.6) be diluted to final concentration 5 μ g/ml, every hole 100 μ l are coated with 96 hole enzyme plates, and 4 ℃ are spent the night.Suck excessive envelope antigen, add 5% skim-milk (described per-cent is mass percent) that people dilutes with PBS, 37 ℃ of sealing 30min; The PBST solution for MAb-B4C2 antibody of purifying (wherein the degree of tween 20 is 0.1%) is respectively to 1:400 by volume, 1:800,1:1600.1:3200,1:6400,1:12800 dilutes, and with every hole 100 μ l, adds in people's enzyme plate; Incubate warm 1h for 37 ℃; With PBST solution, wash away excessive primary antibodie (primary antibodie is herein MAb-B4C2 antibody), add the HRP mark goat anti-mouse igg antibody of people 1:1000 (volume ratio) dilution, every hole 100 μ l, incubate warm 1h for 37 ℃; TMB colour developing, 450nm measures absorbance, represents the power of antigen antibody reaction, and ELISA detected result shows that the titre of gained MAb-B4C2 antibody is up to 1:12000.
The evaluation of the anti-human Fc ε R I α subunit monoclonal antibody of embodiment 2 hybridoma cell strain B4C2 secretions
1. flow cytometry screens hybridoma cell strain positive colony
The CHO3D10 cell of taking the logarithm vegetative period (Chinese hamster ovary cell, Long March hospital laboratory is granted), phosphate buffered saline buffer (PBS) washing, 1000rpm/min, centrifugal 5min; Abandon supernatant liquor, getting bovine serum albumin-phosphate buffered saline buffer (BSA-PBS, described per-cent is mass percent) of 1%, to adjust cell concn be 2 * 10
5individual/ml; Get the anti-human Fc ε R I α subunit monoclonal antibody (embodiment 1 prepares gained) that 1ml CHO3D10 cell adds 100 μ l purifying, hatch 45min for 4 ℃, PBS washing 2 times, centrifugal 1000rpm/min, 5min, abandons supernatant, adds sheep anti-mouse igg (the H+L)-PE fluorescence antibody of 100 μ l working concentrations, hatch 45min for 4 ℃, PBS washing 2 times, centrifugal 1000rpm/min, 5min, abandon supernatant, add 0.5ml PBS to mix rear upper machine testing.
Result shows that the anti-human Fc ε R I α subunit monoclonal antibody of hybridoma secretion with the CHO3D10 cell of expressing people Fc ε R I α subunit, high specific binding can occur and react, the combination rate of this anti-human Fc ε R I α subunit monoclonal antibody and CHO3D10 cell is up to 97.4%, and concrete outcome is asked for an interview shown in Fig. 1 (A) (scattered light result) and Fig. 1 (B) (combination rate result).
2. the anti-human Fc ε of immuno-fluorescence assay R I α subunit monoclonal antibody
The CHO3D10 cell that the phase of taking the logarithm grows, PBS washing, centrifugal 1000rpm/min, 5min; Utilizing 1% BSA-PBS (described per-cent is mass percent) substratum to adjust cell concn is 5 * 10
6individual/ml; Be inoculated in 96 porocyte culture plates, every hole adds 100 μ l cell suspensions; Add paraformaldehyde to fix, every hole adds the anti-human Fc ε of 100 μ l R I α subunit monoclonal antibody, and (the anti-human Fc ε R I α subunit monoclonal antibody adding is that embodiment 1 prepares gained, its concentration is 1.14mg/ml), after ambient temperature overnight, abandon supernatant, PBS washes 2 times; Abandon supernatant, every hole adds sheep anti-mouse igg (H+L) fluorescence antibody of 100 μ l FITC marks, under room temperature, acts on 30min, PBS washing 2 times, fluorescence microscope result.
Result shows: anti-human Fc ε R I α subunit monoclonal antibody MAb-B4C2 can be combined with CHO3D10 cell-specific.
3.Western blot identifies anti-human Fc ε R I α subunit monoclonal antibody
Utilize the prepared albumen of CHO3D10 lysis of expressing people Fc ε R I α subunit, carry out SDS-PAGE electrophoresis, electrotransfer, to PVDF membrane (PVDF) film, through 5% skim-milk sealing 1h, adds 4 ℃ of overnight incubation of anti-human Fc ε R I α subunit monoclonal antibody routinely.The sheep anti-mouse igg antibody incubated at room 1h that adds HRP mark, observations after colour developing.
Result shows that anti-human Fc ε R I α subunit monoclonal antibody MAb-B4C2 presents specific reaction zone to the CHO3D10 cell extract being transferred on pvdf membrane at relative molecular mass 47KD place, and result as shown in Figure 2.
The application of the anti-human Fc ε of embodiment 3 R I α subunit monoclonal antibody MAb-B4C2
The tonsil that derives from tissue is cut into slices and toast 1h in 58 ℃ of baking ovens, put into successively immediately dimethylbenzene I 15min, 15min in dimethylbenzene II take out tissue slice from baking oven after; Tissue slice is soaked one minute successively in dehydrated alcohol, 95% ethanol, 75% ethanolic soln, then wash twice with distillation; Tissue microwave thermal in PH6.0 Citric Acid repair liquid is repaired to 15min, be then cooled to room temperature; By after tissue slice PBS washing three times, get rid of and dry the upper tissue of section liquid around, lie against in wet box, drip the 3% peroxidase blocker hydrogen peroxide of 50 μ l in tissue, lucifuge reaction 20min; After PBS washing three times, get rid of and dry tissue liquid around on tissue slice, drip confining liquid (5%BSA-PBS, described per-cent is mass percent) 50 μ l room temperature 20min; Abandon in tissue after confining liquid, with after PBS washing three times, drip the anti-human Fc ε of 0.7mg/ml R I α subunit monoclonal antibody MAb-B4C2 (embodiment 1 prepares gained) the 250 μ l of 1:200 dilution, 4 ℃ of night incubation; 4 ℃ of tissue slicies that spend the night are removed to unconjugated primary antibodie three times with PBS washing, then add sheep anti-mouse igg-horseradish peroxidase 50 μ l incubated at room 30min; Tissue slice is washed in PBS to three removals unconjugated two anti-, get rid of and dry the upper tissue of section liquid around, lie against in wet box, the DAB nitrite ion (1:50 volume dilution) that adds 50 μ l, under opticmicroscope, control colour developing, slice, thin piece is put into tap water termination reaction when seeing positive findings, and in flowing water, rinsed at least 10min.
Tissue slice is put into after Hematorylin solution 30s, put into distilled water anti-blue, tap water rinses 10min; Tissue slice is soaked to 1min successively in 75% ethanol, 95% ethanol, dehydrated alcohol; After tissue dimethylbenzene is transparent, neutral gum mounting, micro-Microscopic observation, its result is as shown in Figure 3.
Result shows: anti-human Fc ε R I α subunit monoclonal antibody prepared by the present invention can specific binding with the mastocyte in tissue.The anti-human Fc ε R I α subunit monoclonal antibody MAb-B4C2 of hybridoma cell strain B4C2 secretion therefore of the present invention can be used for preparing the detection reagent of people Fc ε R I α subunit expression, for expression qualitative or detection by quantitative cell people Fc ε R I α subunit.
Embodiment 4 hybridoma cell strain B4C2 vitro culture
Get embodiment 1 and prepare gained hybridoma cell strain B4C2, utilize DMEM substratum (buying from Gibco), 37 ℃, cultivate 24 hours, the centrifugal 5min of 3000rpm, collect supernatant liquor, with obtaining anti-human Fc ε R I α subunit monoclonal antibody after NAb Protein G SpinKit (buying from Pierce company) purifying.Through ELISA method described in embodiment 1, measure the anti-human Fc ε of gained R I α subunit monoclonal antibody and tire, the titre of this antibody is: 1:12800, is placed in-70 ℃ of preservations by the anti-human Fc ε of gained R I α subunit monoclonal antibody.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a hybridoma cell strain, is characterized in that, it is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C201211.
2. an anti-human Fc ε R I α subunit monoclonal antibody, is characterized in that, described monoclonal antibody is produced by hybridoma cell strain secretion claimed in claim 1.
3. a preparation method for anti-human Fc ε R I α subunit monoclonal antibody as claimed in claim 2, is characterized in that, described preparation method comprises the following steps:
(1) described in vitro culture claim 1, hybridoma cell strain obtains nutrient solution;
(2), by step (1) gained medium centrifugal, collect supernatant purifying and obtain anti-human Fc ε R I α subunit monoclonal antibody.
4. preparation method as claimed in claim 3, is characterized in that, the temperature of the described cultivation of step (1) is 37 ℃, and the time of cultivation is 24 hours.
5. preparation method as claimed in claim 3, is characterized in that, the described centrifugal speed of step (2) is 3000rpm, and the centrifugal time is 5 minutes.
6. a detection method for cell people Fc ε R I α subunit expression, is characterized in that, this detection method comprises the following steps:
(1) anti-human Fc ε R I α subunit monoclonal antibody described in claim 2 is contacted with separated cell to be detected or the lysate of this cell;
(2) by immunofluorescence analysis method, flow cytometry method, immunoblotting assay method, elisa assay method, immunohistochemical analysis method or immunoprecipitation analysis method, judged whether positive reaction.
7. detection method as claimed in claim 6, is characterized in that, the described cell of step (1) is eukaryotic cell.
8. detection method as claimed in claim 6, is characterized in that, the described immunofluorescence analysis method of step (2) comprises the following steps: the object cell in vegetative period phase of taking the logarithm, PBS washing, centrifugal collecting cell; Cell is inoculated in to Tissue Culture Plate, adds paraformaldehyde to fix, every hole adds anti-human Fc ε R I α subunit monoclonal antibody described in claim 2, and temperature is bathed, and abandons supernatant after washing, and every hole adds fluorescently-labeled dynamics, fluorescence microscope result.
9. detection method as claimed in claim 6, is characterized in that, the described flow cytometry method of step (2) comprises the following steps: the object cell in vegetative period phase of taking the logarithm, PBS washing, centrifugal collecting cell; In cell, add anti-human Fc ε R I α subunit monoclonal antibody described in claim 2, temperature is bathed, and the centrifugal supernatant of abandoning after washing, adds fluorescently-labeled dynamics, upflowing cell instrument detected result.
10. the purposes of anti-human Fc ε R I α subunit monoclonal antibody as claimed in claim 2 in preparing anti-allergy dermatitis, dermatomyositis or psoriasis.
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| CN104792687A (en) * | 2015-04-29 | 2015-07-22 | 辽宁医学院附属第一医院 | Identification method of primed state of basophil |
| CN110577598A (en) * | 2018-06-11 | 2019-12-17 | 李莉 | anti-sFc epsilon RI alpha monoclonal antibody and application thereof |
| WO2019238137A1 (en) * | 2018-06-11 | 2019-12-19 | 李莉 | ANTI-SFCεRIα MONOCLONAL ANTIBODY AND APPLICATION THEREOF |
| CN110577598B (en) * | 2018-06-11 | 2022-03-08 | 李莉 | anti-sFc epsilon RI alpha monoclonal antibody and application thereof |
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