CN107188961A - A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof - Google Patents
A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof Download PDFInfo
- Publication number
- CN107188961A CN107188961A CN201710580686.XA CN201710580686A CN107188961A CN 107188961 A CN107188961 A CN 107188961A CN 201710580686 A CN201710580686 A CN 201710580686A CN 107188961 A CN107188961 A CN 107188961A
- Authority
- CN
- China
- Prior art keywords
- aspergillus flavus
- flavus resisting
- resisting toxin
- monoclonal antibodies
- immunosorbent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a kind of aspergillus flavus resisting toxin M1 monoclonal antibody immunities adsorbent, immune affinity column and preparation method thereof.The anti-AFM1 immunosorbent includes solid phase carrier and the aspergillus flavus resisting toxin M1 monoclonal antibodies being coupled with the solid phase carrier, and the solid phase carrier is the activated resin containing epoxide group；The heavy chain of the aspergillus flavus resisting toxin M1 monoclonal antibodies is IgG2b types, light chain is λ types, 15 amino acid sequences are that 15 amino acid sequences are EVILVESGGGLVKPG before GVTQESALTTSPGGT, the heavy chain aminoterminal before the light chain aminoterminal, and molecular weight is 150KD.The Conjugate ratio of aspergillus flavus resisting toxin M1 monoclonal antibodies of the present invention and activated resin is 93.4%, anti- AFM1 monoclonal antibody immunities adsorbent is 100 ug/mg antibody to the adsorption capacity of Aflatoxins M1 in breast, and the immunosorbent can be applied in fresh milk processing remove Aflatoxins M1 on lossless fresh milk nutrition foundation.
Description
Technical field
The present invention relates to a kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent and preparation method thereof, further relate to
A kind of preparation method for the immune affinity column for preparing the immunosorbent, belongs to technical field of food science.
Background technology
Aflatoxins M1（Aflatoxin M1,AFM1）It is primarily present in breast, therefore is initially referred to as " milk poison
Element ", is published on the Nature magazines of 1964 after the discovery such as de Iongh.With the deep discovery of research, AFM1 is yellow
Aspertoxin B1（Aflatoxin B1,AFB1）Be in vivo through in the presence of Cytochrome P450 serial enzymes through hydroxylating institute shape
Into, it is considered that AFB1 Metabolism of hydroxyl content is the removing toxic substances form of toxin.But AFM1 be not to be seen as be AFB1 removing toxic substances
Product, has many experimental studies to show that AFM1 has cytotoxicity and carcinogenicity, is the weight of current influence dairy products security
Want one of nuisance.
Due to Aflatoxins M1（Abbreviation AFM1）High temperature resistant and stability are strong, in the process of dairy products generally
Can not directly it be removed by high temperature sterilization technique, therefore, there is presently no the dirt that AFM1 in breast is removed by direct technology means
AFM1 measure only has using strict legal residue limits standard and precise detection technology in the method for dye, control breast
OK, once exceeded be accomplished by being destroyed the emulsion for polluting AFM1, huge economic loss is often resulted in.Typical case is exactly
State General Administration for Quality Supervision discloses Mengniu within 2011（Meishan）Co., Ltd and the liquid of Fujian Chang Fu dairy products Co., Ltd production
Contain in body milk and social greatly fear is caused after exceeded AFM1, while also causing huge economic loss to dairy industry company.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of aspergillus flavus resisting toxin M1 monoclonal antibodies, and the antibody has
Very high secretion specificity and affinity, can specifically bind AFM1 in breast.The invention solves the problems that second technical problem
It is to provide a kind of immunosorbent prepared by above-mentioned aspergillus flavus resisting toxin M1 monoclonal antibodies and preparation method thereof.The present invention
The 3rd technical problem to be solved is to provide a kind of immune parent prepared by above-mentioned aspergillus flavus resisting toxin M1 monoclonal antibodies
With post and preparation method thereof.
The technical problem to be solved in the present invention is realized by following scheme：
1st, a kind of aspergillus flavus resisting toxin M1 monoclonal antibodies, the heavy chain of the aspergillus flavus resisting toxin M1 monoclonal antibodies is IgG2b
Type, light chain is λ types, before 15 amino acid sequences are GVTQESALTTSPGGT, the heavy chain aminoterminal before the light chain aminoterminal
15 amino acid sequences are EVILVESGGGLVKPG, and molecular weight is 150KD；The aspergillus flavus resisting toxin M1 monoclonal antibodies with
Aflatoxins M1, aflatoxin M 2, aflatoxin B1, aflatoxin B 2, aflatoxin G 1, aflatoxin
G2, deoxynivalenol and BSA cross reacting rate are respectively 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%,
0%th, 0%, affinity costant is 5.5 × 10-10mol/L.
The preparation method of the Aflatoxins M1 monoclonal antibody, comprises the following steps：
（1）The acquisition of the hybridoma strain of aspergillus flavus resisting toxin M1 monoclonal antibodies
The hybridoma strain is that the spleen cell of gained and myeloma cell after mouse is immunized with comlete antigen AFM1-BSA
SP2/0 fusions are obtained, the active indifference of 30 generation secretory antibodies of passage, and hybridoma can stably excreting in serum free medium
Aspergillus flavus resisting toxin M1 monoclonal antibodies, cultivate resisting to complete cell death wild Oryza species moderate resistance AFM1 in CO2 incubators
Bulk concentration is up to 0.15mg/mL；
（2）The screening of the hybridoma strain of aspergillus flavus resisting toxin M1 monoclonal antibodies
The screening of the aspergillus flavus resisting toxin M1 monoclonal antibody hybridoma cells strain contrasts ELISA method, sieve using high flux
Process is selected to comprise the following steps：
Immunizing potency is selected more than 10-5Mouse spleen cells above carry out supplementary immunization, and extracting spleen cell and SP2/0 are thin after 3 days
Born of the same parents press 1：1 ratio carries out cell fusion in the case where molecular weight is 1450 50%PEG effect, and fused cell cultivates keynote using HAT
It is evenly distributed to after whole cell density in 45 piece of 96 orifice plate, in 5% CO2With 37 DEG C of incubator culture 10 days；Inhaled after 10 days
Supernatant is taken to carry out the screening in positive hole, screening is using in each 45 piece of 96 hole of preprepared BSA and AFM1-BSA coating plates
Plate using equipment liquid relief, the board-washing such as automatic sample injector, Transtar-96, anti-plus nitrite ion of Jia 2, add terminate liquid after use enzyme mark
Instrument determines OD values, and foundation drags hole count and the cell for showing recessiveness in plate is coated with BSA in the aobvious positive that AFM1-BSA is coated with plate
Screening object for the first time is in hole, and each fused cell colony for drawing positive hole respectively using micromanipulative technique is transferred to new 96
Cultivated in well culture plate, be subcloned after culture medium discoloration, obtain the cell strain system of the anti-AFM1 monoclonal antibodies of stably excreting；
（3）The preparation of aspergillus flavus resisting toxin M1 monoclonal antibodies
Using albumin A/G purifying, including step in detail below：
First with 105/ ml initial density is cultivated the hybridoma of screening in 1 liter of revolving bottle, and culture cell is complete
After portion is dead supernatant, buffered liquid 1 are collected using Large Copacity centrifuge：It is pure using albumin A/G purification columns after 1 dilution
Change, collect eluent and concentrate and purify antibody using dialysis membrane of the Amicon stirring-types ultrafiltration apparatus through 30,000 molecular weight, concentration is anti-
Body obtains aspergillus flavus resisting toxin M1 monoclonal antibodies after ND1000 type ultramicron ultraviolet specrophotometer is quantitative；
The hybridoma cell strain of aspergillus flavus resisting toxin M1 monoclonal antibodies tie up to the applying date before document in the public can obtain：
The document is：《Aspergillus flavus resisting toxin M1 monoclonal antibodies are prepared using HTS-ELISA screening techniques》Microorganism journal .ISTIC PKU-
10 Pei's generation spring phase, He Na, Zhang Lijun, Lu Meisheng in 2010.The public can be made according to the method for above-mentioned document；Can also be by
It is made according to the inventive method.
2nd, a kind of aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents and preparation method thereof：
A kind of aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents, the immunosorbent includes solid phase carrier and solid with this
The aspergillus flavus resisting toxin M1 monoclonal antibodies of phase carrier conjugation, the solid phase carrier is the activated resin containing epoxide group.
The preparation method of above-mentioned aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents is：Specifically include following steps：
1）The preparation of polymer microballoon：
Polyvinylpyrrolidone is dissolved in into absolute ethyl alcohol to be added in there-necked flask with deionized water mixed liquor, homogeneous system is stirred into
Afterwards, nitrogen purge is passed through, and rises to required 70 DEG C of reaction temperature；The disposable benzene second added dissolved with initiator azodiisobutyronitrile
Alkene and GMA monomer, the terminating reaction after reaction 24h under stirring and nitrogen protection；By polymerizate
Centrifugal sedimentation is carried out, clear liquid is discarded, absolute ethyl alcohol is added and disperses again, then is settled, it is repeated multiple times, add deionized water again
Scattered, sedimentation is repeated multiple times, is then dried in vacuo, and obtains macromolecule resin；
1）Activated resin：After macromolecule resin is fully swelled with distilled water, drain, it is 2mol/L to add concentration according to 1g/2L
NaOH solution, while stirring plus epoxychloropropane, 24h are reacted at 40 ~ 50 DEG C, after draining, with acetone and distillation water washing tree
Fat, that is, obtain activated resin；
2）Coupling：Activated resin is used to the coupling buffer for being more than 5 times of gel volumes（0.1 mol/L, NaHCO3, pH8.4）Take out
Wash 5 times, be transferred quickly to the coupling buffer containing Aflatoxins M1 monoclonal antibody（0.1 mol/L, NaHCO3,
pH8.4）In, the gentle agitation overnight of room temperature 1h or 4 DEG C makes Aflatoxins M1 monoclonal antibody be fully coupled with activated resin,
Stand, collect binding resin；
3）Closing：Binding resin is added into level pad（0.1 mol/L, pH 8.0, Tris-HCl）, middle room temperature closing 2h；
4）Washing：Then 200mL acetum is used（0.1 mol/L, pH4.0）And level pad（0.1 mol/L,
Tris-HCl, pH8.0）Successively alternately washing 4~6 times, use level pad after washing（0.1 mol/L pH 8.0, Tris-
HCl）Balance, that is, be made aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents.
3. a kind of preparation method for the immune affinity column for loading aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents, tool
Body includes step in detail below：Column jecket is taken, sieve plate has been padded, coupling aspergillus flavus resisting toxin M1 monoclonals prepared by claim 3
The immunosorbent of antibody is fitted into column jecket, instills level pad（0.1 mol/L, pH 8.0, Tris-HCl）Make glue surface steady
It is fixed, seal a mouthful bottom.
The beneficial effects of the invention are as follows：Aspergillus flavus resisting toxin M1 monoclonal antibodies of the present invention are a kind of high with secretion
The aspergillus flavus resisting toxin M1 monoclonal antibodies of specificity and high-affinity, the aspergillus flavus resisting toxin M1 monoclonal antibodies are bent with Huang
It is mould toxin M1, aflatoxin M 2, aflatoxin B1, aflatoxin B 2, aflatoxin G 1, aflatoxin G 2, de-
Oxygen nivalenol and BSA cross reacting rate are respectively 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%, 0%,
Affinity costant is 5.5 × 10-10mol/L.Utilize specific the aspergillus flavus resisting toxin M1 monoclonal antibodies and special carrier material system
Standby immunosorbent of the present invention is the idol of efficiently single-minded extra corporeal immunoadsorption agent, anti-AFMI monoclonal antibodies and activated resin
Connection rate is 93.4%, and the adsorption capacity of Aflatoxins M1 is 100 ug/mg antibody, and the immunosorbent can be applied to fresh milk and add
Aflatoxins M1 is removed in work on lossless fresh milk nutrition foundation, and then ensures the security of dairy products.
Brief description of the drawings
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
Fig. 1 is the monoclonal antibody SDS-PAGE pictures in embodiment 3.
Fig. 2 is the moderate resistance AFM of embodiment 41The identification picture of monoclonal antibody subclass.
Fig. 3 is different toxin in embodiment 5 to AFM1The inhibiting rate curve of monoclonal antibody.
Fig. 4 is the moderate resistance AFM of embodiment 61Monoclonal antibody affinity qualification result curve.
Embodiment
（1）Material and instrument
Main agents, consumptive material and laboratory apparatus are as shown in table 1 needed for present invention experiment.
（2）Immune animal and cell
SP2/0 cells are purchased to univ cambridge uk MRC, and BABL/C mouse are purchased from Harbin veterinary institute.
Reagent consumptive material and instrument and equipment needed for table 1 is tested
| Reagent and instrument | Source |
| Aflatoxins M1, M2, B1, B2, G1, G2 | Sigma companies |
| Aflatoxin M1-BSA holoantigens | Sigma companies |
| Bovine serum albumin(BSA) BSA, ovalbumin OVA | Sigma companies |
| Freund's complete adjuvant and incomplete Freund's adjuvant | Sigma companies |
| Milipore filter (30,000NMWL, diameter 44.5mm) | Millipore, Ultracel YM-30 |
| HAT and HT culture mediums | Sigma companies |
| PEG | Sigma companies |
| Mouse monoclonal antibody typing detection reagent | Sigma companies |
| MD6 serum free mediums | Grand celebration Mai Baikang Bioisystech Co., Ltd |
| NBCS, hyclone | Hyclone companies |
| 96 hole elisa Plates and Tissue Culture Plate | Costar companies |
| Ultramicron ultraviolet-uisible spectrophotometer | GE companies, NanoVue |
| Microwell plate knockout | BIOTEK companies of the UFILL types U.S. |
| ELIASA | BIOTEK companies of the ELX808 types U.S. |
| Full-automatic sample-adding machine | Genetix companies of Britain QFill3 |
| 96 hole pipettors | Corning companies |
| Biological reactor for cell culture | Dutch Applikon |
| Board-washing machine | SIM companies of the U.S. |
| CO2Incubator | SIM companies of the U.S. |
| Biohazard Safety Equipment | SIM companies of the U.S. |
Embodiment 1
A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies, the heavy chain of the aspergillus flavus resisting toxin M1 monoclonal antibodies is IgG2b types,
Light chain is λ types, and 15 amino acid sequences are 15 before GVTQESALTTSPGGT, the heavy chain aminoterminal before the light chain aminoterminal
Individual amino acid sequence is EVILVESGGGLVKPG, and molecular weight is 150KD；The aspergillus flavus resisting toxin M1 monoclonal antibodies and Huang
Aspertoxin M1, aflatoxin M 2, aflatoxin B1, aflatoxin B 2, aflatoxin G 1, aflatoxin G 2,
Deoxynivalenol and BSA cross reacting rate are respectively 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%,
0%, affinity costant is 5.5 × 10-10mol/L.
The preparation process of above-mentioned aspergillus flavus resisting toxin M1 monoclonal antibodies and identification are as follows：
1st, animal immune scheme
It is immunized from 86 week old BABL/C mouse, immunogene is complete A antigen FM1-BSA, each immunizing dose is 20 μ
Only, subcutaneous and intraperitoneal injection is immunized once g/ every two weeks.Initial immunity emulsifies comlete antigen, 2-3 times using Freund's complete adjuvant
It is immune that comlete antigen is emulsified using incomplete Freund's adjuvant.Dock after 3rd time immune 10 days blood sampling, detected with indirect elisa method
Serum titer.
Use coating buffer（Carbonate buffer solution）AFM1-BSA is diluted to after 0.1 μ g/ml concentration, wrapped by every μ l/ holes of hole 100
By ELISA Plate, cleaning solution is used after ambient temperature overnight（PBS containing 0.05% Tween-20）Board-washing 3 times.Plus confining liquid（1% bovine serum albumin
Liquid）100 μ l/ holes, 37 DEG C of incubation 1h, are washed 3 times.After blood serum sample doubling dilution to be checked, 100 μ l/ holes, 37 DEG C of incubation 1h are washed
Wash 3 times.ELIAS secondary antibody is with 1：100 μ l/ holes are added after 5000 times of dilutions, 37 DEG C of incubation 1h are washed 3 times.Add tmb substrate solution
100 μ l/ holes, lucifuge stands 10min, plus terminate liquid（2M H2SO4）50 μ l/ holes, OD values at 450nm are determined using ELIASA.When
Highest serum extension rate when OD values are more than or equal to 2 times of negative control OD value is serum titer.3rd immunized mice blood
Clear mean titre can reach 1：0.5x105More than.
2nd, cell fusion, screening and cloning approach
（1）The preparation of SP2/O myeloma cell：
36-48h will cultivate benefit and be cultivated in myeloma cell's expansion of exponential phase before fusion, will with elbow straw before fusion
Cell is blown down from cell culture bottle wall, is collected in 50ml centrifuge tubes, and 1200rpm is centrifuged 4 minutes, abandons supernatant.Add
20ml1640 nutrient solutions, are mixed again, and 1200 rpm are centrifuged 4 minutes, abandon supernatant, plus 10ml1640 nutrient solutions, are mixed.Take cell
Suspension 0.1ml, adds 0.9ml1% platforms and expects orchid, mixes, and drips in being counted under microscope on tally, standby.
（2）The preparation of splenocyte：
Select antiserum titre 1：0.5x105More than BABL/C mouse in merging preceding 3 days abdominal cavity booster immunizations 1 time, immunogene
Only, adjuvant is not added with for AFM1-BSA, 20 ug/.Pluck eyeball of mouse bloodletting before fusion, cervical dislocation is put to death, 75% alcohol-pickled disappears
Mouse, is then fixed on the mouse plate of Biohazard Safety Equipment by poison.Sterile opening abdominal cavity, takes out spleen, is trained in containing 10ml 1640
Washed twice in the plate of nutrient solution, remove the adipose tissue and connective tissue of surrounding, be placed in the flat of the nutrient solution containing 10ml 1640
In ware.200 mesh mesh screens are taken to be put in plate, plus spleen is placed on mesh screen in nutrient solution by 1640 nutrient solutions to mesh screen is submerged,
Spleen is gently extruded with L-type rod, makes splenocyte completely into nutrient solution.Splenocyte liquid is sucked in 50ml centrifuge tubes,
1400rpm is centrifuged 4 minutes, abandons supernatant, then adds 10ml1640 nutrient solutions, is mixed.Ibid cell count, it is standby.
（3）Cell fusion：
It is 8.47 × 10 to take Mouse spleen cells count results7, after being merged with myeloma cell, using adding 1%HAT and 10%
To assign to 45 piece of 96 hole flat thin in 280uL/ holes after FBS 1200mL MD6 mammalian cells serum free medium dilution fused cell
In born of the same parents' culture plate, in 5% CO2 With closing culture 10-12 days in 37 DEG C of incubator.Moved after 10-12 days using Transtar-96
It is preprepared with Tissue Culture Plate phase that liquid device takes the μ L/ holes of supernatant 50 to be transferred to respectively respectively in thing bacterium clean bench
It is corresponding and be coated with AFM1-BSA and BSA each 45 pieces of elisa plates, utilize high-flux detection method screening positive hybridoma
Cell hole.
（4）The screening of positive colony and clone：
The screening in positive hole is carried out after 10 days in Aspirate supernatant, screening is using in preprepared BSA and AFM1-BSA bags
Added and be incubated after supernatant respectively by each 45 piece of 96 orifice plate of plate, the board-washing based on classical indirect ELISA, Jia 2 anti-plus develops the color
OD values are determined with ELIASA after liquid plus terminate liquid, select in BSA coating plates to show in the aobvious positive that AFM1-BSA is coated with plate
Recessive cell hole is screening object for the first time, draws each fused cell colony in positive hole respectively using micromanipulative technique
It is transferred in 96 new well culture plates and cultivates, Confirm-ELISA is carried out after culture medium discoloration, collects the positive cell strain confirmed and enter
Row culture, takes the hole cell supernatant of single clone to carry out antibody test, testing result continues to expand culture for positive clone
After freeze and for preparing antibody.Its result is as shown in table 2.
The positive colony hybridoma that table 2 is obtained
3rd, the preparation and purification scheme of aspergillus flavus resisting toxin M1 monoclonal antibodies
（1）Serum free medium prepares antibody：
The positive cell strain that will confirm that, which expands to be transferred to after culture in 1 liter of rolling bottle, cultivates 14 days, collects 1 after medium centrifugal：1 with
PBS mixed albumin A post, was eluted with pH3.7 eluent, and 3 are utilized by Amicon stirring-type ultrafiltration apparatus
The milipore filter of ten thousand molecular weight is concentrated, and concentrate dispenses laggard after being quantified using the ultramicron ultraviolet specrophotometer of GE companies
Row is freeze-dried and preserved.
（2）Mouse ascites method prepares ascites antibody：
Take the healthy Balb/C mouse in 10 week old postpartum, intraperitoneal injection 500 μ L sterilizing paraffin oils, mouse peritoneal injection after seven days
106Hybridoma/only, after mouse web portion swelling after 7 days, collect ascites and purify.
（3）SDS-PAGE:
Anti- AFM1 monoclonal antibodies prepared by the anti-AFM1 monoclonal antibodies prepared by ascites method, Serium-free Culture, and nothing after purification
The monoclonal antibody of serum free culture system carries out SDS-PAGE respectively, as a result as shown in figure 1, the monoclonal antibody foreign protein prepared by the visible ascites method of the figure
It is more, and then purity is higher for the antibody of free serum culture, purification effect is good.
4th, the qualification program of aspergillus flavus resisting toxin M1 monoclonal antibody subclass
Kit is determined according to SBA Clonotyping System/HRP antibody subtypes, the hybridization for secreting anti-AFM1 monoclonal antibodies is utilized
Oncocyte supernatant carries out subgroup identification.Method is：By coating antigen AFM1-BSA by 1,0.5,0.25mg/L coated elisa plates, 100 μ
L/ holes, 4 DEG C overnight；After 1.5% BSA closings, by monoclonal antibody, gradient dilution is laggard in the ranks connects non-competing ELISA inspection since 1mg/L
Survey.Its result is as shown in Figure 2, it is seen that obtained anti-AFM1 monoclonal antibodies heavy chain is that IgG2b types, light chain are λ chains, antibody light chain aminoterminal
Preceding 15 amino acid sequences are GVTQESALTTSPGGT, and 15 amino acid sequences are before heavy chain aminoterminal
EVILVESGGGLVKPG, molecular weight is 150KD.
5th, between aspergillus flavus resisting toxin M1 monoclonal antibody mabs and other toxin cross reacting rate measure side
Case
With coating antigen AFM1-BSA coated elisa plates, 100ul/ holes, 4 DEG C overnight, are poured out liquid in hole, cleaning solution（Told containing 0.05%
The PBS liquid of temperature 20）Washing 3 times.Plus confining liquid（1% bovine serum albumin liquid）100ul/ holes, 37 DEG C of incubation 1h, pour out liquid in hole
Body, is washed 3 times.Take different diluted concentrations（1000ng/ml、100ng/ml、10ng/ml、1ng/ml、0.1ng/ml、0.01ng/
ml、0.001ug/ml、0.0001 ug/ml）Aflatoxins M1, aflatoxin M 2, aflatoxin B1, aspergillus flavus poison
Plain B2, aflatoxin G 1, aflatoxin G 2, deoxynivalenol and BSA standard items and isometric appropriate concentration
（0.1 ug/ml）Monoclonal antibody mixing be added in coating plate, 100ul/ holes, 37 DEG C of incubation 1h pour out liquid in hole, washing three
It is secondary.Again plus ELIAS secondary antibody（1：2000 times of dilutions）, 100ul/ holes, 37 DEG C of incubation 1h, washing 3 times.Add tmb substrate solution
150ul/ holes, lucifuge stands 10min, plus terminate liquid（2M H2SO4）50ul/ holes, the suction in each hole at 450nm is determined using ELISA Plate
Light rate（A）.According to formula：Competitive assays rate=（The A values of Positive control wells A values-blocking aperture）/ Positive control wells A values * 100% is counted
Competitive assays rate is calculated, further according to formula：Competitor when AFM1 concentration/inhibiting rate is 50% when cross reacting rate=inhibiting rate is 50%
Concentration * 100% calculates cross reacting rate.As a result as shown in figure 4, antibody and Aflatoxins M1, aflatoxin M 2, aspergillus flavus
Toxin B1, aflatoxin B 2, aflatoxin G 1, aflatoxin G 2, deoxynivalenol and BSA intersection are anti-
Should rate be respectively 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%, 0%.
6th, the measure scheme of aspergillus flavus resisting toxin M1 monoclonal antibody affinity
By coating antigen AFM1-BSA respectively with 1,0.5 and 0.25ug/ml concentration coated elisa plate, 100ul/ holes, 37 DEG C of incubations
1h, pours out liquid in hole, uses cleaning solution（PBS liquid containing 0.05% polysorbas20）3 of washing add multiple proportions and are serially diluted（1：
400、1：1600、1：6400、1：25600、1：102400、1：409600、1：1638400）Monoclonal antibody, 100ul/ holes,
37 DEG C of incubation 1h, pour out liquid in hole, wash 3 times.Plus ELIAS secondary antibody（1：1000 times of dilutions）100ul/ holes, 37 DEG C of incubation 1h,
Washing 3 times.Tmb substrate solution 100ul/ holes are added, lucifuge stands 10min, plus terminate liquid（2M H2SO4）50ul/ holes, using enzyme
Target determines the absorptance in each hole at 450nm（A）.With antibody concentration（Ab）For abscissa, light absorption value is that ordinate draws three
The curve map of difference coating concentration, as shown in Figure 4.The 50% of 3 curves is found out respectivelyODAntibody concentration point corresponding to max
It is not 1.12 × 10-11mol/L、1.26×10-11mol/L、1.35×10-11Mol/L, according to formula Kaff=(n-1)/2(n
[Ab '] t- [Ab] t) obtains 3 K altogetheraffValue is 4.0 × 10 respectively10 M-1、3.0×1010 M-1、9.0×1010M-1, take it to put down
The affinity costant that average is monoclonal antibody is 5.5 × 1010M-1。
Embodiment 2
A kind of aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents, the immunosorbent includes solid phase carrier and solid with this
The aspergillus flavus resisting toxin M1 monoclonal antibodies of phase carrier conjugation, the solid phase carrier is the activated resin containing epoxide group.
The preparation method of above-mentioned aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents is：Specifically include following steps：
1）The preparation of polymer microballoon：
Polyvinylpyrrolidone is dissolved in into absolute ethyl alcohol to be added in there-necked flask with deionized water mixed liquor, homogeneous system is stirred into
Afterwards, nitrogen purge is passed through, and rises to required 70 DEG C of reaction temperature；The disposable benzene second added dissolved with initiator azodiisobutyronitrile
Alkene and GMA monomer, the terminating reaction after reaction 24h under stirring and nitrogen protection；By polymerizate
Centrifugal sedimentation is carried out, clear liquid is discarded, absolute ethyl alcohol is added and disperses again, then is settled, it is repeated multiple times, add deionized water again
Scattered, sedimentation is repeated multiple times, is then dried in vacuo, and obtains macromolecule resin；
1）Activated resin：After macromolecule resin is fully swelled with distilled water, drain, it is 2mol/L to add concentration according to 1g/2L
NaOH solution, while stirring plus epoxychloropropane, 24h are reacted at 40 ~ 50 DEG C, after draining, with acetone and distillation water washing tree
Fat, that is, obtain activated resin；
2）Coupling：Activated resin is used to the coupling buffer for being more than 5 times of gel volumes（0.1 mol/L, NaHCO3, pH8.4）Take out
Wash 5 times, be transferred quickly to the coupling buffer containing Aflatoxins M1 monoclonal antibody（0.1 mol/L, NaHCO3,
pH8.4）In, the gentle agitation overnight of room temperature 1h or 4 DEG C makes Aflatoxins M1 monoclonal antibody be fully coupled with activated resin,
Stand, collect binding resin；
3）Closing：Binding resin is added into level pad（0.1 mol/L, pH 8.0, Tris-HCl）, middle room temperature closing 2h；
4）Washing：Then 200mL acetum is used（0.1 mol/L, pH4.0）And level pad（0.1 mol/L,
Tris-HCl, pH8.0）Successively alternately washing 4~6 times, use level pad after washing（0.1 mol/L pH 8.0, Tris-
HCl）Balance, that is, be made aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents.
Embodiment 3
A kind of immune affinity column for being mounted with aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents, its preparation method include with
Lower specific steps：Column jecket is taken, sieve plate has been padded, coupling aspergillus flavus resisting toxin M1 monoclonal antibodies prepared by claim 3 are exempted from
Epidemic disease adsorbent is fitted into column jecket, instills level pad（0.1 mol/L, pH 8.0, Tris-HCl）Make glue surface stable, seal mouth
Bottom.
The absorption property of the immunosorbent of embodiment 4 determines scheme
All there are 1mg aspergillus flavus resistings by coupling added with the mL of 100ng, 200ng, 300ng, 400ng AFM1 fresh milks solution 500
The immune affinity column of the immunosorbent of toxin M1 monoclonal antibodies, each 3 repetitions of concentration, washing, elution, collection elution
AFM1 contents in liquid, IC-ELISA detection collection liquids, last Computation immunity adsorption capacity adds AFM1 contents as shown in Table 3
In below 200ng, adsorption rate is more than 90%, and adsorption capacity is close to 100 ug/mg antibody.
The maximal absorptive capacity of the AFM1 monoclonal antibody immunities of table 3 parent's adsorbent
| Addition (ng) | Make the adsorption rate of immunosorbent by oneself（%） |
| 100 | 93.1 |
| 200 | 92.6 |
| 300 | 81.5 |
Claims (4)
1. a kind of aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents, it is characterised in that：The immunosorbent includes solid
Phase carrier and the aspergillus flavus resisting toxin M1 monoclonal antibodies being coupled with the solid phase carrier, the solid phase carrier are to contain epoxide group
Activated resin；The heavy chain of the aspergillus flavus resisting toxin M1 monoclonal antibodies is IgG2b types, and light chain is λ types, the light chain amino
Hold preceding 15 amino acid sequences for GVTQESALTTSPGGT, 15 amino acid sequences are before the heavy chain aminoterminal
EVILVESGGGLVKPG, molecular weight is 150KD.
2. a kind of method for preparing immunosorbent as claimed in claim 1, it is characterised in that：Specifically include following steps：
1）The preparation of polymer microballoon：
Polyvinylpyrrolidone is dissolved in into absolute ethyl alcohol to be added in there-necked flask with deionized water mixed liquor, homogeneous system is stirred into
Afterwards, nitrogen purge is passed through, and rises to required 70 DEG C of reaction temperature；The disposable benzene second added dissolved with initiator azodiisobutyronitrile
Alkene and GMA monomer, the terminating reaction after reaction 24h under stirring and nitrogen protection；By polymerizate
Centrifugal sedimentation is carried out, clear liquid is discarded, absolute ethyl alcohol is added and disperses again, then is settled, it is repeated multiple times, add deionized water again
Scattered, sedimentation is repeated multiple times, is then dried in vacuo, and obtains macromolecule resin；
1）Activated resin：After macromolecule resin is fully swelled with distilled water, drain, it is 2mol/L to add concentration according to 1g/2L
NaOH solution, while stirring plus epoxychloropropane, 24h are reacted at 40 ~ 50 DEG C, after draining, with acetone and distillation water washing tree
Fat, that is, obtain activated resin；
2）Coupling：Activated resin is used to the coupling buffer for being more than 5 times of gel volumes（0.1 mol/L, NaHCO3, pH8.4）Take out
Wash 5 times, be transferred quickly to the coupling buffer containing Aflatoxins M1 monoclonal antibody（0.1 mol/L, NaHCO3,
pH8.4）In, the gentle agitation overnight of room temperature 1h or 4 DEG C makes Aflatoxins M1 monoclonal antibody be fully coupled with activated resin,
Stand, collect binding resin；
3）Closing：Binding resin is added into level pad（0.1 mol/L, pH 8.0, Tris-HCl）, middle room temperature closing 2h；
4）Washing：Then 200mL acetum is used（0.1 mol/L, pH4.0）And level pad（0.1 mol/L,
Tris-HCl, pH8.0）Successively alternately washing 4~6 times, use level pad after washing（0.1 mol/L pH 8.0, Tris-
HCl）Balance, that is, be made aspergillus flavus resisting toxin M1 monoclonal antibody immunity adsorbents.
3. a kind of immune affinity column for the immunosorbent being mounted with described in claim 2.
4. a kind of method for preparing immune affinity column as claimed in claim 3, it is characterised in that：Including step in detail below：
Column jecket is taken, sieve plate has been padded, the immunosorbent of coupling aspergillus flavus resisting toxin M1 monoclonal antibodies prepared by claim 2 loads
In column jecket, level pad is instilled（0.1 mol/L, pH 8.0, Tris-HCl）Make glue surface stable, seal a mouthful bottom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710580686.XA CN107188961A (en) | 2017-07-17 | 2017-07-17 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710580686.XA CN107188961A (en) | 2017-07-17 | 2017-07-17 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107188961A true CN107188961A (en) | 2017-09-22 |
Family
ID=59882550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710580686.XA Pending CN107188961A (en) | 2017-07-17 | 2017-07-17 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107188961A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386980A (en) * | 2019-07-16 | 2019-10-29 | 河南检通生物技术有限公司 | A kind of aspergillus flavus-resistance mycin G1(AFG1) method for preparing monoclonal antibody |
| CN111389053A (en) * | 2020-04-07 | 2020-07-10 | 柳家鹏 | Immunoadsorbent, device and preparation method for purifying mouse monoclonal antibody in hybridoma cell culture solution |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160472A (en) * | 2011-12-09 | 2013-06-19 | 北京中检维康技术有限公司 | Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit |
| CN103215230A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit |
| CN204594992U (en) * | 2015-04-21 | 2015-08-26 | 齐齐哈尔大学 | Aflatoxins M1 absorption affinity column pick-up unit in milk |
| CN105566494A (en) * | 2016-01-26 | 2016-05-11 | 华中农业大学 | Monoclonal antibody and enzyme-linked immunosorbent assay method for detecting aflatoxin M1 |
-
2017
- 2017-07-17 CN CN201710580686.XA patent/CN107188961A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160472A (en) * | 2011-12-09 | 2013-06-19 | 北京中检维康技术有限公司 | Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit |
| CN103215230A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit |
| CN204594992U (en) * | 2015-04-21 | 2015-08-26 | 齐齐哈尔大学 | Aflatoxins M1 absorption affinity column pick-up unit in milk |
| CN105566494A (en) * | 2016-01-26 | 2016-05-11 | 华中农业大学 | Monoclonal antibody and enzyme-linked immunosorbent assay method for detecting aflatoxin M1 |
Non-Patent Citations (3)
| Title |
|---|
| 何娜: "基于HTS-ELISA的抗黄曲霉毒素M_1单克隆抗体制备新方法研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 张小舟等: "高分子微球免疫吸附剂的制备及其对黄曲霉毒素M1的吸附性能", 《食品科学》 * |
| 李湛勇等: "单克隆抗体免疫吸附剂的制备及对乙型肝炎表面抗原的吸附", 《高分子学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386980A (en) * | 2019-07-16 | 2019-10-29 | 河南检通生物技术有限公司 | A kind of aspergillus flavus-resistance mycin G1(AFG1) method for preparing monoclonal antibody |
| CN111389053A (en) * | 2020-04-07 | 2020-07-10 | 柳家鹏 | Immunoadsorbent, device and preparation method for purifying mouse monoclonal antibody in hybridoma cell culture solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101831407B (en) | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof | |
| US20150276729A1 (en) | Immunosorbent and immunoaffinity column for aflatoxin m1 nanobody and preparation method thereof | |
| CN105112375B (en) | Hybridoma cell strain ZJED0-02, anti-Ebola virus GP protein monoclonal antibody and its preparation and application | |
| Conant et al. | Rhinoviruses: Basis for a Numbering System: 1. HeLa Cells for Propagation and Serologic Procedures | |
| CN101825633A (en) | Competitive ELISA kit for detecting african swine fever virus antibody and purposes thereof | |
| CN107459574A (en) | A kind of PRV gB monoclonal antibodies and its application | |
| CN104877968B (en) | Efficient secretion anti-dog parvovirus monoclonal antibody hybridoma cell A135 strains | |
| CN107188961A (en) | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof | |
| CN108699137A (en) | Target the antibody of the O- antigens of the Friedlander's bacillus based on mannosan | |
| CN104560886B (en) | One plant of anti-strain of natamycin monoclonal antibody hybridoma cell and its application | |
| CN101793897A (en) | Method for improving screening efficiency of hybridoma | |
| CN108103002A (en) | Preparation of mdck cell host's residual protein and application thereof | |
| CN108752471A (en) | The preparation method and applications of anti-PCV2 monoclonal antibodies | |
| CN105255838B (en) | Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies | |
| CN101671655B (en) | Monoclonal antibody hybridoma cell of HIV P24 and application | |
| CN105349496B (en) | The hybridoma cell strain of anti-macroglobulin monoclonal antibody | |
| CN106544324B (en) | Monoclonal antibody of mycoplasma hyopneumoniae and application thereof | |
| CN112011517B (en) | Panda LBP monoclonal antibody hybridoma cell strain and application thereof | |
| CN104789535B (en) | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application | |
| CN105349497B (en) | Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody | |
| CN108192868A (en) | The induced amplification method of immunocyte | |
| JP5467228B2 (en) | Rapid detection method of Campylobacter in stool | |
| CN107083370A (en) | Anti-treeing Shrew interferon gammas monoclonal antibody and hybridoma cell strain and the application for secreting the antibody | |
| KR100207351B1 (en) | Antibody against human plasmin-alpha 2-plasmin inhibitor complex, hybridoma and immunoassay | |
| CN106520704A (en) | Anti-quinolone antibiotic class specific monoclonal antibody hybridoma cell strain YH6 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170922 |