CN101906159A - Hybridoma cell line D6D - Google Patents
Hybridoma cell line D6D Download PDFInfo
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- CN101906159A CN101906159A CN 201010204412 CN201010204412A CN101906159A CN 101906159 A CN101906159 A CN 101906159A CN 201010204412 CN201010204412 CN 201010204412 CN 201010204412 A CN201010204412 A CN 201010204412A CN 101906159 A CN101906159 A CN 101906159A
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Abstract
The invention relates to the development of a hybridoma cell line D6D, and belongs to the field of biotechnology. OTA-BSA obtained by coupling BSA and OTA is used to immunize a BALB/c mice; splenic cells of the mice are fused with Sp2/0 myeloma cells; and the fused cells are selectively cultured by an HAT culture medium. A complete antigen OTA-KLH obtained by coupling KLH and OTA is taken as a coating antigen of ELISA; antibody content in hybridoma cell culture supernatant is detected by an indirect ELISA method; the hybridoma cell line D6D stably secreting anti-OTA monoclonal antibodies is screened; the detected D6D ascite ELISA titer is 1:3,200; identification shows that the monoclonal antibody can be specifically combined with the OTA, and an SDS-PAG electrophoretogram of the purified monoclonal antibody has two protein bands which are respectively about 50 KD and 25 KD and coincide with the molecular weight magnitudes of a light chain and a heavy chain denatured from an antibody protein, so that the fact that an anti-OTA specific antibody is obtained is proved again, the subtype of the monoclonal antibody is IgG2b, and the light chain is a kappa chain. The specific monoclonal antibody can be used for researching the OTA and establishing an OTA immunological detection method.
Description
Technical field
(Ochratoxin A, OTA) hybridoma cell line D 6 D of monoclonal antibody belongs to biological technical field, relates to the antibody engineering technology to the present invention relates to secrete ochratoxin A.
Background technology
Ochratoxin (Ochratoxins) is that a class is mainly by aspergillus fungi (for example, Aspergillus ochraceus AspergillusOchraceus, sulfuraspergillus Aspergillus Fresenii etc.) and the mycetogenetic mycotoxins of Penicillium.OTA is the main ingredient of ochracin, also is the major cause that causes ochracin to be poisoned, and it urgees carcinous and carinogenicity is confirmed in rat.Epidemiology survey finds that OTA may have certain dependency with area, human Balkan ephrosis to the pollution of grain products.IARC (IARC) is divided into 2B class carcinogens to OTA.
The structure of OTA is the phenylformic acid Isocoumarin 〉97.It is a kind of stable colourless crystallization compound, is slightly acidic, is dissolved in polar solvent and sodium hydrogen carbonate solution, is slightly soluble in water, and its methanol solution is preserved in refrigerator and can not be decomposed in 1 year.OTA molecular formula C
20H
18ClNO
6, molecular weight is 404, is a kind of haptens material.
The chemical structural formula of OTA
OTA extensively is present in agricultural-food such as cereal such as wheat, barley, oat, corn and coffee berry, grape and the goods, and human health and animal husbandry development are constituted potential threat.(The CodexCommittee on Food Additives and Contaminants, CCFAC) etc. international organization thinks that OTA is a kind of mycotoxins of the public health security that counts for much for the foodstuff additive and the pollutent code council.The limit standard of OTA is formulated in the 56th the FAO/WHO foodstuff additive joint specialist council (JECFA) meeting, thinks in cereal such as wheat, barley and rye and the goods thereof that it is 5 μ gkg that OTA limits the quantity of
-1
Harm in view of OTA, by preparing its monoclonal antibody, the foundation that can be research of ochratoxin antigenicity and OTA immunological detection method provides necessary component and technique means, to the biological function of exploring each structural region of OTA and set up OTA special, the sensitive detection method is significant.
Summary of the invention
Technical problem
The hybridoma cell strain that the purpose of this invention is to provide secretion OTA monoclonal antibody.Monoclonal antibody of the present invention is that bovine serum albumin (BSA) is prepared complete antigen as carrier proteins and OTA coupling, and immune BALB/c mouse utilizes hybridoma technology to obtain the monoclonal antibody D6D of energy specific recognition OTA.
Technical scheme
A kind of OTA monoclonal antibody is characterized in that: this monoclonal antibody is that hybridoma cell line D 6 D produces, and hybridoma cell line D 6 D is preserved in Chinese typical culture collection center on May 27th, 2010, and deposit number is CCTCC NO:C201052.The preparation method comprises:
1) preparation of immunizing antigen OTA-BSA
Dimethyl sulfoxide (DMSO) (DMSO) solution of preparation OTA adds N-hydroxyl succinic diamide (NHS) and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and carries out the activation of OTA.Above-mentioned solution slowly dripped in bovine serum albumin (BSA) solution carry out the coupling of OTA and BSA, reaction product obtains the conjugate of OTA and carrier proteins with phosphate buffered saline buffer (PBS) dialysis.
2) mouse immune
Low dose of macrocyclic immunization protocol is adopted in this experiment.Use coupling protein OTA-BSA as immunizing antigen, the female BALB/c mouse (available from Yangzhou University comparative medicine center) in 7 ages in week is carried out immunity.By 200 μ g/ immunizing dose only BALB/c mouse is carried out 8 immunity, last immunity was won mouse spleen after three days, did cell and merged.
3) cytogamy
Get the BALB/c mouse splenocyte uniform mixing in proportion of Sp2/0 myeloma cell's (be so kind as to give the academy of agricultural sciences in Jiangsu Province) and immunity; under 50% Macrogol 4000 (PEG 4000) effect, carry out cytogamy, use 1640 substratum (available from Gibico company) that contain 20%FCS (purchasing the biological scientific and technological Graduate School of Engineering of intelligent people) and HAT (purchasing company) at 5%CO in Invitrogen in Shanghai
2Cultivate in the incubator, after cultivating 14d, progressively use 1640 substratum of 20%FCS and HT (purchasing company) and 1640 culture medium culturing of 20%FCS in Invitrogen, when the cell that merges grow to 96 orifice bore floorages 1/5 the time, get supernatant and carry out anti-OTA monoclonal antibody and detect.
4) hybridoma screening
Carbonate buffer solution with OTA-hemocyanin (KLH) is done coating antigen, does confining liquid with 1% gelatin, and 1: 10000 horseradish peroxidase target sheep anti-mouse igg is as two anti-(available from doctor's moral biotech companies), TMB-H
2O
2Be substrate, 2mol L
-1Sulphuric acid soln is a stop buffer, PBST does washing lotion, the negative reference serum of the positive reference serum of dilution in 1: 800 and dilution in 1: 800 is done the indirect enzyme-linked immunosorbent assay (ELISA) of contrast, anti-OTA antibody-secreting situation in the hybridoma supernatant is detected the positive hybridoma cell of the anti-OTA antibody of screening secretion.Measure OD through microplate reader
450nmValue: with the blank zeroing, as the OD of positive reference serum
450nmThe OD of value and negative reference serum
450nmRatio 〉=2.1 of value are detected the hole and are judged to the positive, as the OD of positive reference serum
450nmThe OD of value and negative reference serum
450nmThe detection hole of the ratio of value＜1.5 is judged to feminine gender, and ratio is judged to suspicious between the centre.
5) limiting dilution assay is to the hybridoma subclone
At first positive hole viable cell is expected that with platform orchid dyes and count, be diluted to 100 cells/15mL substratum, the cell suspension of dilution is added 96 porocyte culture plates, reach 1 cell in every hole, at 37 ℃, 5%CO with 1640 perfect mediums
2Cultivate in the incubator.Get cell conditioned medium when cultivating 8～9d, in time carry out ELISA and detect.Select the male monoclonal cell to carry out same subclone again more than 3 times, detect all positive and to detect OD value more approaching in each hole, with the positive cell enlarged culturing after the subclone cultivation repeatedly and frozen until this cell institute foraminous supernatant liquor.
6) anti-OTA MONOCLONAL ANTIBODIES SPECIFIC FOR
With 5 * 10 of PBS dilution
6～1 * 10
7Individual positive hybridoma cell injects the BALB/c mouse abdominal cavity of growing up, and gathers belly behind 5～10d and obviously heaves the ascites of mouse, and is centrifugal, collects supernatant.
7) evaluation-denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of anti-OTA monoclonal antibody purity
The monoclonal antibody ascites solution of anti-OTA mixes with 5 * sample-loading buffer, sex change is boiled in water-bath, through denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and coomassie brilliant blue R250 dyeing, according to electrophoretic antibody protein band quality size, the existence of checking antibody.
(6) evaluation of antibody subtype
(Rache Iso strip, cat.NO.14932027) antagonism OTA monoclonal antibody is carried out the hypotype test with Mouse Monoclonal Antibody Isotyping Kit.
Beneficial effect characteristics of the present invention and advantage are as follows:
1, antigen prepd method provided by the invention is simple and feasible.
2, OTA monoclonal antibody high specificity provided by the invention, the preparation method is simple.
3, monoclonal antibody specific provided by the invention can be used for the research of OTA and the foundation of OTA immunological detection method.
4, obtain the hybridoma cell line D 6 D of stably excreting OTA monoclonal antibody, recording that D6D ascites ELISA tires is 1: 3200.Through identifying that this monoclonal antibody is the IgG2b antibody-like, light chain of antibody is the κ hypotype.
Description of drawings
The continuous wave spectrum scanning of Fig. 1 coupled antigen ultraviolet detects
The electrophorogram of monoclonal antibody behind Fig. 2 purifying
Biological preservation
Hybridoma cell line D 6 D is preserved in Chinese typical culture collection center, address: Wuhan Wuhan University postcode on May 27th, 2010: 430072, and deposit number is CCTCC C201052.
Embodiment
(1) preparation of immunizing antigen OTA-BSA and detection
The activation of OTA: get 1mg OTA and be dissolved in the 0.25mL DMSO solution, measure NHS and EDC respectively and add (OTA: NSH: the EDC mol ratio is 1: 2: 4) in the above-mentioned solution, lucifuge is acutely shaken 2h under the room temperature, and 4 ℃ of reactions are afterwards spent the night.The coupling of OTA: above-mentioned solution dripped slowly (2mg albumen is dissolved in 1mL 0.1mol L in BSA solution
-1In the sodium hydrogen carbonate solution), add the magnetic force rotor, place on the magnetic force vibrator lucifuge vibration 2h under the room temperature; Reaction product is in 0.05mol L
-14 ℃ of following lucifuge dialysis 72h in the PBS damping fluid, every 5h changes dialyzate once, obtains the conjugate of OTA and carrier proteins.
The complete antigen that coupling is good is verified by the ultraviolet continuous sweep method.Configuration OTA standardized solution, BSA standardized solution and coupling complete antigen diluent.The BSA standardized solution maximum absorption band occurs at 280nm, and the OTA standardized solution maximum absorption band occurs at 379nm.In 200～400nm wavelength, 5nb carries out continuous wavelength scanning at interval.Result such as Fig. 1.
(2) foundation of hybridoma cell strain
1) mouse immune
Low dose of macrocyclic immunization protocol is adopted in this experiment.Use coupling protein OTA-BSA as immunizing antigen, the female BALB/c mouse in 7 ages in week is carried out immunity.By 200 μ g/ immunizing dose only, 1: 1 (V/V) mixes with Freund's complete adjuvant, and emulsification 30min reaches the water-in-oil shape, does not dissolve in water or 4 ℃ of not stratified effective emulsifications that are considered as of spending the night.With the emulsive complete antigen, nape portion multiple spot subcutaneous injection immunity BALB/c mouse (200 μ g/ are only); After 2 weeks, with same antigen and isopyknic Freund's incomplete adjuvant emulsification, 200 μ g/ only carry out booster immunization; After 7 immunity, do not add adjuvant and only carry out the abdominal injection booster immunization with 200 μ g/.From for the third time, each one week of immunity back tail vein or eyeball blood sampling, 37 ℃ leave standstill 1h, 4 ℃ are spent the night and separate out serum, 3000r min
-1Centrifugal 10min, separation of serum test sera titre.Last immunity was won mouse spleen after three days, did cell and merged.
2) cytogamy
The BALB/c mouse splenocyte of getting Sp2/0 myeloma cell and immunity by 1: 5～1: 10 mixed in the 50mL centrifuge tube, abundant mixing, the centrifugal 10min of 1000rpm, abandon supernatant, touch the pipe end with palm, make cell loose evenly, put 40 ℃ of water-bath preheatings, add the 1mL 50%PEG 4000 that is preheated to 40 ℃ with the 1mL suction pipe in 45s, the limit edged vibrates gently, adds 30mL then and be preheated to 37 ℃ 1640 incomplete substratum in 90s, room temperature leaves standstill 10 min, the centrifugal 10min of 1000rpm abandons supernatant, and adding 20mL, to contain 1640 substratum of 20%FCS and HAT resuspended.Divide on 96 orifice plates that install to existing feeder cell, in 5%CO
2Incubator is cultivated, and behind the 7d, the upgrowth situation of observation of cell is with 1640 substratum that contain 20%FCS and HT, 1/2 substratum that swaps out; Behind the 14d, use 20%FCS and HT 1640 culture medium culturing instead, when the cell that merges grow to 96 orifice bore floorages 1/5 the time, get supernatant and carry out antibody test.
3) anti-OTA hybridoma screening
With coating buffer is 0.05mol L
-1PH 9.6 carbonate buffer solutions, the envelope antigen that coupling is good (OTA-KLH) is made the doubling dilution bag by elisa plate, and 100 μ L/ holes are put 4 ℃ of bags and are spent the night; PBST washing 3 times, each 5min pats dry for the last time; Seal every hole with 1% gelatin, 2h is placed for 37 ℃ in 300 μ L/ holes; PBST washing 3 times, each 5min pats dry for the last time; With the positive reference serum of cell conditioned medium, dilution in 1: 800 and the negative reference serum of dilution in 1: 800, add in the respective aperture 100 μ L/ holes, 37 ℃ of effect 60min; PBST washing 3 times, each 5min pats dry for the last time; Add the enzyme mark sheep anti-mouse igg of dilution in 1: 10000,60min is placed for 37 ℃ in 100 μ L/ holes; PBST washing 5 times, each 5min pats dry for the last time; Add substrate TMB-H
2O
2, 100 μ L/ holes, room temperature lucifuge colour developing 15min; Every hole adds 100 μ L 2mol L
-1The sulphuric acid soln termination reaction.Measure OD through microplate reader
450nmValue: with the blank zeroing, as the OD of positive reference serum
450nmThe OD of value and negative reference serum
450nmRatio 〉=2.1 of value are detected the hole and are judged to the positive, as the OD of positive reference serum
450nmThe OD of value and negative reference serum
450nmThe detection hole of the ratio of value＜1.5 is judged to feminine gender, and ratio is judged to suspicious between the centre.
4) limiting dilution assay carries out subclone to positive hybridoma cell
Positive hybridoma cell for the secretion monoclonal antibody specific that screens gained in time adopts limiting dilution assay to carry out subclone.At first positive hole viable cell is expected that with platform orchid dyes and count, be diluted to 100 cells/15mL substratum with 1640 perfect mediums, the cell suspension of dilution is added 96 porocyte culture plates, the 0.15mL/ hole is at 37 ℃, 5%CO
2Cultivate in the incubator.Behind 4～5d, microscopically can be observed the formation of clone cell, and record has only single clonal growth hole, gets cell conditioned medium during 8～9d, in time carries out ELISA and detects.Select the male monoclonal cell to carry out same subclone again more than 3 times, detect all positive and to detect OD value more approaching in each hole until all cells hole supernatant liquor; With the positive cell enlarged culturing after repeatedly subclone is cultivated and frozen, obtain the hybridoma cell strain called after D6D of stably excreting OTA monoclonal antibody.
(3) ascites of the anti-OTA monoclonal antibody of production
With the adult BALB/c mouse of paraffin oil injection, 0.5mL/, sensitization is after 7 days, with positive hybridoma cell 0.01mol L
-1Mouse peritoneal, every injected in mice 5 * 10 are injected in PBS dilution back
6～1 * 10
7, 0.2mL takes belly obviously to heave the ascites of mouse behind 5～10d, 10000rpm, and centrifugal 5min collects supernatant, packing in a small amount ,-70 ℃ of preservations.
(4) the ascites titration of anti-OTA monoclonal antibody
With PBS damping fluid dilution in 1: 100 monoclonal antibody ascites, carry out doubling dilution then, add the enzyme plate of KLH-OTA bag quilt, 60min, is hatched in 100 μ L/ holes by 37 ℃; PBST washing 3 times, each 5min pats dry for the last time; Add the HRP mark sheep anti-mouse igg of dilution in 1: 10000,60min, is hatched in 100 μ L/ holes by 37 ℃; PBST washing 5 times, each 5min pats dry for the last time; Add substrate TMB-H
2O
2, 100 μ L/ holes, room temperature lucifuge colour developing 15min; Every hole adds 100 μ L 2mol L
-1The sulfuric acid termination reaction; Measure OD with microplate reader
450nmValue.With negative OD
450nmValue detects hole and negative OD less than 0.2
450nmThe ratio of value is judged to the positive greater than 2.1, tires as the ascites of monoclonal antibody with the greatest dilution in positive hole.The result shows that D6D cell strain ascites indirect ELISA titer is 1: 3200.
(5) evaluation one denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of anti-OTA monoclonal antibody purity
Preparation 10mL 12% separation gel solution.Between two sheet glass, pour into separation gel rapidly, use water-lute above.Behind about 30min, the water on rubber cover upper strata is poured out in the complete polymerization of glue to be separated, blots with filter paper.Preparation 5% concentrates glue, is poured on the separation gel, inserts comb afterwards immediately, careful bubble.After waiting to concentrate gelling admittedly, carefully extract comb, with electrophoresis buffer solution for cleaning hole.Sample and 5 * sample-loading buffer are mixed in proportion, and heated and boiled 10min is centrifugal in the water, the about 20 μ L of sample on every hole.Fill it up with electrophoretic buffer, the 80V electrophoresis, when sample was pressed into straight line in concentrated glue after, regulating voltage arrived separation gel bottom, powered-down to 120V up to tetrabromophenol sulfonphthalein.Unload lower glass plate, knock sheet glass open, Yi Bian after marking, scrape gel with spatula.Coomassie brilliant blue R250 dye liquor dyeing 2h.Decolouring is spent the night high-visible to band, takes pictures, as Fig. 2.
(6) evaluation of antibody subtype
Test with Mouse Monoclonal Antibody Isotyping Kit.At first ascites is diluted with PBS, to antibody concentration be 0.1～1 μ gmL
-1, get 150 μ L and add testing tube.15～25 ℃, 30s shakes the vortex pipe then, and the interior blue colloid of pipe is fully suspended, and blackhead inserts test strip down, behind 5～10min, with reference to positive control, blue band place occurs and is this antibody subtype classification.The hypotype of this monoclonal antibody is IgG2b, and light chain is the κ chain.
Claims (2)
1. OTA monoclonal antibody is characterized in that: this monoclonal antibody is that hybridoma cell line D 6 D produces, hybridoma cell line D 6 D on May 27th, 2010 precious deposits in China typical culture collection center, deposit number is CCTCC NO:C201052.
2. a kind of OTA monoclonal antibody according to claim 1 is characterized in that, is obtained by following method preparation:
1) preparation of immunizing antigen OTA-BSA
Get 1mg OTA and be dissolved in the 0.25mLDMSO solution, add NHS and EDC, lucifuge is acutely shaken 2h under the room temperature, and 4 ℃ of reactions are spent the night again, and above-mentioned solution is slowly dripped the solution in BSA, lucifuge vibration 2h under the room temperature, and reaction product is in 0.05mol L
-1Lucifuge dialysis 72h in the PBS damping fluid obtains the conjugate of OTA and carrier proteins;
2) mouse immune
Low dose of macrocyclic immunization protocol is adopted in this experiment, use coupling protein OTA-BSA as immunizing antigen, female BALB/c mouse to 7 ages in week is carried out immunity, by 200 μ g/ immunizing dose only, mixed in 1: 1 by volume with Freund's complete adjuvant, emulsification reaches the water-in-oil shape, with the emulsive complete antigen, and nape portion multiple spot subcutaneous injection immunity BALB/c mouse, 200 μ g/ only, after 2 weeks, with same antigen and isopyknic Freund's incomplete adjuvant emulsification, 200 μ g/ only carry out booster immunization, after 7 immunity, do not add adjuvant and only carry out the abdominal injection booster immunization with 200 μ g/, last immunity was won mouse spleen after three days, did cell and merged;
3) cytogamy
It is even by 1: 5～1: 10 mixed with the BALB/c mouse splenocyte of immunity to get Sp2/0 myeloma cell, the centrifugal 10min of 1000rpm, abandon supernatant, touch the pipe end with palm, make cell loose evenly, put 40 ℃ of water-bath preheatings, in 45s, add the 1mL 50%PEG 4000 that is preheated to 40 ℃ with the 1mL suction pipe, the limit edged vibrates gently, adds 30mL then and be preheated to 37 ℃ 1640 incomplete substratum in 90s, and room temperature leaves standstill 10min, the centrifugal 10min of 1000rpm, abandon supernatant, adding 20mL, to contain 1640 substratum of 20%FCS and HAT resuspended, divides on 96 orifice plates that install to existing feeder cell, cultivate in the 5%CO2 incubator, behind the 7d, the upgrowth situation of observation of cell is with 1640 substratum that contain 20%FCS and HT, 1/2 substratum that swaps out, behind the 14d, use 20%FCS and HT 1640 culture medium culturing instead, when the cell that merges grow to 96 orifice bore floorages 1/5 the time, get supernatant and carry out antibody test;
4) hybridoma of the anti-OTA antibody of screening secretion
With coating buffer is 0.05mol L
-1PH 9.6 carbonate buffer solutions, the envelope antigen OTA-KLH that coupling is good makees the doubling dilution bag by elisa plate, 100 μ L/ holes are put 4 ℃ of bags and are spent the night, PBST washing 3 times, each 5min pats dry for the last time, seals every hole with 1% gelatin, 2h is placed for 37 ℃ in 300 μ L/ holes, PBST washing 3 times, each 5min, pat dry for the last time, with cell conditioned medium, the negative reference serum of the positive reference serum of dilution in 1: 800 and dilution in 1: 800 adds in the respective aperture, 100 μ L/ holes, 37 ℃ of effect 60min, PBST washing 3 times, each 5min, pat dry for the last time, the sheep anti-mouse igg that adds the horseradish peroxidase-labeled of dilution in 1: 10000,60min is placed for 37 ℃ in 100 μ L/ holes, PBST washing 5 times, each 5min pats dry for the last time, adds substrate TMB-H
2O
2, 100 μ L/ holes, room temperature lucifuge colour developing 15min, every hole adds 100 μ L 2mol L
-1The sulphuric acid soln termination reaction is measured OD through microplate reader
450nmValue is judged positive hybridoma cell, and positive hybridoma cell is carried out the hybridoma cell strain called after D6D that subclone obtains the anti-ochratoxin A monoclonal antibody of stably excreting through limiting dilution assay;
5) hypotype of anti-OTA monoclonal antibody
Test with Mouse Monoclonal Antibody Isotyping Kit, this anti-OTA monoclonal antibody hypotype is IgG2b, and light chain is the κ chain.
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| CN105349524A (en) * | 2015-11-17 | 2016-02-24 | 清华大学 | Hybridomas cell strain of anti-human coagulation factor VII polyclonal antibody |
| CN105400769A (en) * | 2015-11-17 | 2016-03-16 | 清华大学 | Hybridoma cell strain for anticoagulation factor VIII monoclonal antibody |
| CN109535256A (en) * | 2018-12-12 | 2019-03-29 | 深圳市金阅科技有限责任公司 | Application of the ochratoxin A antiidiotype nano antibody as ochratoxin A standard items substitute |
| CN109535251A (en) * | 2018-12-13 | 2019-03-29 | 中国农业科学院油料作物研究所 | A kind of ochratoxin A antiidiotype nano antibody and preparation method thereof |
| CN109575138A (en) * | 2018-12-20 | 2019-04-05 | 中国农业科学院油料作物研究所 | A kind of application of ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute |
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2010
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| 《中国兽医科技》 19931225 张志东等 分泌抗赭曲霉素A单克隆抗体杂交瘤细胞株的建立 , 第12期 2 * |
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Cited By (10)
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| CN105349524A (en) * | 2015-11-17 | 2016-02-24 | 清华大学 | Hybridomas cell strain of anti-human coagulation factor VII polyclonal antibody |
| CN105400769A (en) * | 2015-11-17 | 2016-03-16 | 清华大学 | Hybridoma cell strain for anticoagulation factor VIII monoclonal antibody |
| CN105349524B (en) * | 2015-11-17 | 2019-02-22 | 清华大学 | The hybridoma cell strain of anticoagulin VII polyclonal antibody |
| CN105400769B (en) * | 2015-11-17 | 2019-04-12 | 清华大学 | The hybridoma cell strain of anticoagulin VIII monoclonal antibody |
| CN110408597A (en) * | 2018-12-10 | 2019-11-05 | 浙江工商大学 | One plant of hybridoma cell strain for secreting preventing from heavy metal cadmium ion monoclonal antibody and its application |
| CN109535256A (en) * | 2018-12-12 | 2019-03-29 | 深圳市金阅科技有限责任公司 | Application of the ochratoxin A antiidiotype nano antibody as ochratoxin A standard items substitute |
| CN109535256B (en) * | 2018-12-12 | 2022-10-21 | 深圳市金阅科技有限责任公司 | Application of ochratoxin A anti-idiotype nano antibody as substitute of ochratoxin A standard substance |
| CN109535251A (en) * | 2018-12-13 | 2019-03-29 | 中国农业科学院油料作物研究所 | A kind of ochratoxin A antiidiotype nano antibody and preparation method thereof |
| CN109575138A (en) * | 2018-12-20 | 2019-04-05 | 中国农业科学院油料作物研究所 | A kind of application of ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute |
| CN109575138B (en) * | 2018-12-20 | 2022-08-05 | 中国农业科学院油料作物研究所 | Application of ochratoxin A anti-idiotype nano antibody as ochratoxin A antigen substitute |
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