CN104762267B - Hybridoma AFB1 2A4 and its caused aflatoxin B1 monoclonal antibody - Google Patents
Hybridoma AFB1 2A4 and its caused aflatoxin B1 monoclonal antibody Download PDFInfo
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- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title claims abstract description 30
- 229930020125 aflatoxin-B1 Natural products 0.000 title claims abstract description 28
- 239000002115 aflatoxin B1 Substances 0.000 title claims abstract description 27
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 23
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 title 1
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 title 1
- 241000228197 Aspergillus flavus Species 0.000 claims abstract description 15
- JKKCSFJSULZNDN-UHFFFAOYSA-N gonyautoxin v Chemical compound N=C1NC(COC(=O)NS(O)(=O)=O)C2NC(=N)NC22C(O)(O)CCN21 JKKCSFJSULZNDN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000035945 sensitivity Effects 0.000 claims abstract description 9
- 230000028327 secretion Effects 0.000 claims abstract description 3
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
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- 238000012216 screening Methods 0.000 claims description 7
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- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 abstract description 9
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Abstract
The invention provides aspergillus flavus resisting toxin B1 monoclonal antibodies and its application caused by hybridoma cell strain 2A4, its secretion.Hybridoma cell strain 2A4 can be used for preparing high-titer aflatoxin antibody, and the potency that mouse hydroperitoneum antibody of RGDV Enzyme Linked Immunoadsorbent Assay (ELISA) method measures is up to 3.12 × 106；The aspergillus flavus resisting toxin B1 monoclonal antibody high sensitivities, 50% inhibition concentration to aflatoxin B1 are that IC50 is 33.2pg/mL, and it is used to determine aflatoxin B1 content, and actual application value is big.
Description
Technical field
The present invention relates to hybridoma cell strain AFB1-2A4 and its caused aflatoxin B1 monoclonal antibody.
Background technology
Aflatoxin (Aflatoxin, AF) is a kind of extremely strong extremely toxic substance, is ground by the cancer of the World Health Organization
It is 1 class carcinogenic substance to study carefully mechanism to delimit.Aflatoxin is the metabolite of toxigenic bacterium strain in aspergillus flavus and aspergillus parasiticus, is generally deposited
In the grain, feed and its product that go mouldy.Aflatoxin B1, B2, G1 and G2 are aflatoxin in grain and food
Form is primarily present, the wherein toxicity of aflatoxin B1 and carcinogenicity is most strong.After aflatoxin B1 enters human body, its is main
Target organ is liver, acute poisoning symptom be Nausea and vomiting, stomach and intestine massive haemorrhage and it is dead.Slow poisoning can mainly cause liver cancer,
Osteocarcinoma, kidney, carcinoma of the rectum etc. can also be induced.
At present, detecting the conventional instrumental method of aflatoxin has thin-layered chromatography, high performance liquid chromatography, microtrabeculae chromatography
Method.The degree of accuracy of these methods measure is higher, and repeatability preferably, but often needs to be equipped with large-scale equipment costly, costly, right
Operating personnel require high, are typically adapted to apply in country level research institute or R＆D institution's large-scale experiment room.It is enzyme-linked to exempt from
Epidemic disease method the set high specific of antigen-antibody reaction and the high susceptibility of enzymatic reaction.This method high sensitivity, than thin
Layer method improves nearly 200 times；High specificity, fluorescent material, pigment, analogue are noiseless to result；And the rate of recovery is high,
Extracting method is simple, can carry out qualitative, quantitative measure.Any one that foundation is studied for aflatoxin B1 (AFB1) is exempted from
Epidemiology detection technique, it is necessary to first obtain aspergillus flavus resisting toxin B1 (AFB1) antibody.
The content of the invention
Problem to be solved by this invention is to provide hybridoma cell strain AFB1-2A4 and its caused aflatoxin B1
(AFB1) monoclonal antibody
The invention provides the thin strain AFB1-2A4 of hybridoma, it is micro- that the cell line has been preserved in China on December 12nd, 2014
Biological inoculum preservation administration committee common micro-organisms center (CGMCC), preservation address are China, the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC NO.10200, Classification And Nomenclature S/P20
Hybridoma.
Hybridoma cell strain AFB1- of the aspergillus flavus resisting toxin B1 monoclonal antibodies by deposit number for CGMCC NO.10200
2A4 secretions produce.The AFB1 monoclonal antibodies can identify aflatoxin B1, to 50% inhibition concentration of aflatoxin B1
IC50 is 33.2pg/mL.
Application of the aspergillus flavus resisting toxin B1 monoclonal antibodies in aflatoxin B1 measure.
Hybridoma cell strain AFB1-2A4 monoclonal antibodies provided by the invention are screened using three-step approach and obtained, and it has
Body step is：Generation AFB1 oximes are acted in the hydrochloride of carboxymethyl azanol half (CMO) with AFB1, under the conditions of existing for EDC and NHS
Generation AFB1-BSA is coupled with BSA.BLAB/c mouse are immunized 4 times with it, last time booster immunization 2 times and preceding primary immune response
The AFB1-BSA of dosage, cell fusion is carried out after immune 3 days.Using ELISA, method carries out screening fused cell in two steps：The first step
Filtered out using indirect elisa method and be only capable of positive colonies of the aspergillus flavus resisting toxin B1 without anti-carrier protein BSA；Second step uses
The positive colony nutrient solution that indirect competitive ELISA method filters out to the first step detects, using AFB1 as competition source, selection
Absorbance is low, positive colony in higher sensitivity carries out limiting dilution assay and continues to clone, and clone is after 10 days using same two
Step screening method carries out antibody test, is so repeated 2 times, the hybridoma AFB1-2A4 that final screening obtains.
The preparation method of aflatoxin B1 monoclonal antibody provided by the invention, step are as follows：By the hybridoma of acquisition
The BALB/c mouse that cell line AFB1-2A4 injections are treated with incomplete Freund's adjuvant in advance, collects the ascites of the mouse, pure
Aspergillus flavus resisting toxin B1 monoclonal antibody is obtained after change processing.
The concrete operations of above-mentioned purification process are：Ascites 3000r/min centrifugation more than 5min, draws supernatant in 4 DEG C, will
Supernatant mixes with the acetate buffer of 2 times of volumes, is slowly added to caprylic acid, the caprylic acid needed for every milliliter of ascites while stirring
Volume is 33 μ L, mixed at room temperature 30min, 4 DEG C of standing more than 2h, it is fully precipitated.Then 4 DEG C, 12000r/min centrifugations
More than 30min, precipitation is abandoned, after obtained supernatant is filtered with sand core funnel, add the 0.1mol/L of 1/10 filtrate volume
PH7.4 phosphate buffer, pH to 7.4 is adjusted with 2mol/L sodium hydroxide solution, 4 DEG C of precoolings 1 hour, be slowly added to
For 0.277g/mL ammonium sulfate to ammonium sulfate final concentration of 45%, 4 DEG C stand more than 2h, then 4 DEG C of 12000r/min centrifugations
30min, supernatant is abandoned, gained precipitation is resuspended with the 0.01mol/L phosphate buffers of former ascites volume 1/10, loads dialysis
Bag, dialysed with 0.01mol/L phosphate buffers, the protein solution fully dialysed is added into isometric glycerine, puts -20 DEG C of ice
It is standby in case；
Acetate buffer：0.29g sodium acetates add 0.141mL acetic acid, and pure water is settled to 100mL；
0.1mol/L phosphate buffers：0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride,
0.02g potassium dihydrogen phosphates, add water to be settled to 100mL.
0.01mol/L phosphate buffers：0.1mol/L phosphate buffers are diluted 10 times with pure water.
The beneficial effects of the present invention are：
1 hybridoma AFB1-2A4 provided by the invention can be used for the aspergillus flavus resisting toxin B1 Dan Ke for preparing high-titer
Grand antibody, the potency that aflatoxin B1 mouse ascites antibody ELISA immuno absorbence method determines is reached 3.12 × 106, i.e. abdomen
Water is diluted to 3.12 × 106When solution measurement result be still the positive.
2 aflatoxin B1 monoclonal antibody high sensitivities provided by the invention, specificity are good, to aflatoxin B1
503nhibiting concentration IC50 is 33.2pg/mL, the cross reacting rate 2.3% with aflatoxin B 2, the friendship with aflatoxin G 1
Pitch reactivity 9.9%, the cross reacting rate 0.5% with aflatoxin G 2, the cross reacting rate with Aflatoxins M1
1.6%.
3 aspergillus flavus resisting toxin B1 provided by the invention monoclonal antibody can be used for the content of measure aflatoxin B1.
Brief description of the drawings
Examination criteria curve of Fig. 1 AFB1-2A4 monoclonal antibodies to AFB1；
Embodiment
Embodiment 1：Hybridoma cell strain AFB1-2A4 screening
1. the synthesis of antigen and being immunized for animal
AFB1 obtains AFB1 oximes, then coupling occurs instead with carrier protein under the conditions of existing for EDC and NHS by oximate
Should, obtain corresponding artificial antigen.
The synthesis of AFB1 oximes
1.5 milligrams of AFB1 and 3mg CMO is dissolved in 2mL methanol-pyridine (1: 1) solution, 70 DEG C are reacted 5 hours.Instead
Should after reaction solution is volatilized, with distilled water dissolution residual substance.The pH to 3.0 of solution is adjusted, AFB1O is extracted with 3mL ethyl acetate
3 times.Combined ethyl acetate layer, is volatilized, molten with 0.5mL DMF weights.Obtain AFB1 oxime solution.
The synthesis of artificial antigen
0.45mL AFB1O are taken, add 30mg EDC and 8.64mg NHS, add 1.5mL DMF, 30 DEG C of reactions are overnight.
Reaction solution and the 12.5mg BSA of 3mL 0.1M PBS dissolvings react 2 hours at room temperature.After the completion of reaction, 3000rpm centrifugations 10
Minute, supernatant 2L PBSs 3 times, every time one day.Finally the liquid in bag filter is collected, -20 DEG C of preservations.
The identification of artificial antigen
Show that the complete A antigen FB1- of aflatoxin B1 has been prepared by uv scan method and Mass Spectrometric Identification
BSA。
6 week old female BAl BIcs/c mouse 6 are bought, the complete A antigen FB1- of the immune aflatoxin B1 voluntarily synthesized
BSA.It is immunized for the first time by complete A antigen FB1-BSA and equivalent Freund's complete adjuvant mixing and emulsifying, then mouse nape part is subcutaneous
Multi-point injection.It is immune to after head exempts from 3 weeks and carries out for the second time, using incomplete Freund's adjuvant and isometric complete A antigen FB1-BSA
Emulsification, immunizing dose is exempted from identical with method with head.Third time is immunized and second of 3 weeks immunization interval time, immunizing dose and side
Method is identical with second.Be immune to for 4th time third time it is immune 3 weeks after carry out, immunization wayses and immunizing dose and third time are immune
Identical, each immunizing dose is per the μ g of mouse 30.Latter week is 3 times immunized every time, eye circumference blood sampling, serum is separated, using indirect
ELISA method monitors mice serum antibody titer.3 days after the 4th is immune, tail vein blood, serum is separated, using indirect ELISA
Method monitors mice serum antibody titer, and determines mice serum sensitivity with indirect competitive ELISA method, selects potency, sensitivity
Corresponding to of a relatively high serum mouse carry out last time booster immunization, immunizing dose be before 2 times.
2. cell fusion
After last time booster immunization 3 days, using the polyethylene glycol (PEG, molecular weight 1460) of 50% (percentage by weight)
Make fusion agent, carry out cell fusion according to a conventional method.Comprise the following steps that：
BALB/c mouse is taken off into neck to put to death, is soaked in 75% alcohol.Super-clean bench is put into after about 3min.Lifted with tweezers small
Mouse skin of abdomen, an osculum (attention does not break peritonaeum) is cut with operating scissors, then tears skin to head, tail both direction with tweezers
Skin, fully expose peritonaeum.Use clean scissors instead and cut off peritonaeum, take out spleen, be put into and filled the flat of the endless full nutrient solutions of 10mL
Cleaned once in ware, and peel off connective tissue.Spleen is transferred in another plate for filling the endless full nutrient solutions of 5mL, takes a bronze medal
Net, spleen is placed on copper mesh, spleen is ground with plunger, and copper mesh is rinsed with the endless full nutrient solution in plate, made
Splenocyte is all entered in solution by mesh.Spleen cell solutions are transferred in 50ml centrifuge tubes, add endless full nutrient solution extremely
30mL, mix.1000rpm centrifuges 5min, abandons supernatant.Sedimentation cell cannots be used up full nutrient solution centrifugation once again, then by cell
It is suspended from 10ml complete culture solutions, mixes.Cell count, it is standby.
Myeloma cell and splenocyte are mixed in 1: 10 ratio, washed 1 time with DMEM culture mediums in 50mL centrifuge tubes,
1200rpm centrifuges 8min；Supernatant is abandoned, with suction pipe exhaustion residual liquid.Merged with 50%PEG, with containing 20% cow's serum, 1%HAT
DMEM culture mediums are resuspended.Above-mentioned cell is added in 96 orifice plates of existing feeder layer, adds 100 μ L per hole.Culture plate is put
37 DEG C, cultivate in 5%CO2 incubators.DMEM culture mediums are the DMEM bases culture containing 2% (percetage by weight) growth factor
Base, DMEM basal mediums are purchased from Hyclone companies, and (hypoxanthine-aminopterin-thymidine is purchased from 1%HAT
Gibco companies.
3. screening and the clone of cell line
The 10th day or so after cell fusion, cell colony, which is grown to, accounts for the size of bottom hole 1/2, you can carries out antibody inspection
Survey.The culture hole for having Growth of Hybridoma Cell is screened using ELISA method, screening is carried out in two steps, and the first step uses
Indirect elisa method filters out positive holes of the aspergillus flavus resisting toxin B1 without anti-carrier protein BSA；Second step uses indirect competition
The positive hole that ELISA method filters out to the first step is detected, former by the use of aflatoxin B1 as competition, selects light absorption value and spirit
The higher hole of sensitivity (light absorption value it is higher refer to competition be originally 0 the hole i.e. final tested volume of negative control hole it is higher, sensitivity compared with
Height refer to inhibiting rate for 50% when competition original content that is, IC 50 be worth it is smaller), using limiting dilution assay by hybridoma cell clone
Change, detected using same two-step method within 10 days or so after clone, after such repeated cloning 2 times, obtain hybridoma cell strain
AFB1-2A4。
Embodiment 2：Preparation, purifying and the identification of antibody
The hybridoma cell strain AFB1-2A4 by acquisition that embodiment 1 is obtained is injected in advance with incomplete Freund's adjuvant
The BALB/c mouse managed, the ascites of the mouse is collected, aspergillus flavus resisting toxin B1 monoclonal antibody is obtained after purification process.
Concrete operations are：Ascites centrifuges more than 5min in 4 DEG C of 3000r/min, draws supernatant, by supernatant and 2 times of volumes
Acetate buffer mixes, and is slowly added to caprylic acid while stirring, and the caprylic acid volume needed for every milliliter of ascites is 33 μ L, room temperature
30min is mixed, 4 DEG C of standing 2h, it is fully precipitated.Then 4 DEG C of 12000r/min centrifugation 30min, abandon precipitation, by what is obtained
After supernatant is filtered with sand core funnel, the 0.1mol/L pH7.4 of 1/10 filtrate volume phosphate buffer is added, uses 2mol/
L sodium hydroxide solution regulation pH to 7.4,4 DEG C of precoolings 1 hour, is slowly added to 0.277g/mL ammonium sulfate to ammonium sulfate final concentration
For 45%, 4 DEG C of standing 2h, 4 DEG C of 12000r/min centrifuge 30min, abandon supernatant, by the former ascites volume 1/10 of gained precipitation
0.01mol/L phosphate buffers are resuspended, and load bag filter, are dialysed, will fully dialysed with 0.01mol/L phosphate buffers
Protein solution add isometric glycerine, put standby in -20 DEG C of refrigerators；
Acetate buffer：0.29g sodium acetates add 0.141mL acetic acid, and pure water is settled to 100mL；
0.1mol/L phosphate buffers：0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride,
0.02g potassium dihydrogen phosphates, add water to be settled to 100mL.
0.01mol/L phosphate buffers：0.1mol/L phosphate buffers are diluted 10 times with pure water.
The potency for the mouse hydroperitoneum antibody of RGDV for measuring 2A4 with conventional non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method is reachable
3.12×106, i.e. ascites is diluted to 3.12 × 106When measurement result for the positive.Identify that its is right with conventional indirect competitive ELISA method
The 503nhibiting concentration IC50 of aflatoxin B1 is 33.2pg/mL, the cross reacting rate 2.3% with aflatoxin B 2, with Huang
Aspertoxin G1 cross reacting rate 9.9%, the cross reacting rate 0.5% with aflatoxin G 2, with Aflatoxins M1
Cross reacting rate 1.6%.
Embodiment 3：The application of antibody
The aspergillus flavus resisting toxin B1 monoclonal antibodies that hybridoma cell strain AFB1-2A4 secretes are used for aflatoxin B1
Monoclonal ELISA adds recovery test, comprises the following steps that：
(1) it is coated with：Using 96 hole polystyrene ELISA Plates, with 0.05mol/L pH 9.6 carbonic acid buffer by AFB1-
BSA is diluted to 1 μ g/mL, 100 μ L is added into per hole with the adjustable liquid-transfering gun of multiple tracks (the abbreviation volley of rifle fire), by ELISA Plate after adding
Covered with preservative film, be placed in 4 DEG C overnight.
(2) board-washing：Second day, by ELISA Plate from 4 DEG C of taking-ups, washed using PBST, per the μ l of hole 250, wash 4 times, wash
Patted dry afterwards on towel.
(3) close：200 μ L 1%BSA PBS is added into per hole with the volley of rifle fire, ELISA Plate is covered with lid after adding, put
2h is placed in 37 DEG C.Washed using PBST, per the μ l of hole 250, wash 4 times, patted dry after washing on towel.
(4) 0,0.005,0.015,0.045,0.14,0.41 μ g/L aflatoxin B1 standard is prepared with 10% methanol
Solution.Standard liquid and detected sample extract solution are separately added into the ELISA Plate closed, 50 μ L/ holes, each sample
3 repeating holes of product, add and be diluted to the μ L/ holes of 1: 160000 aflatoxin B1 monoclonal antibody 50, addition is diluted to 1:
The μ L/ holes of 4000 ELIAS secondary antibody 50, gently react 40min in 37 DEG C of insulating boxs after beating ELISA Plate.
(5) board-washing：Operation is same as above.
(6) develop the color：Nitrite ion A and nitrite ion B is mixed by 1: 1 volume ratio, the nitrite ion mixed is added with the volley of rifle fire
100 μ L/ holes, put 37 DEG C of insulating box light protected environment reaction 10min.
(7) determine：2mol/L H are added using the volley of rifle fire2SO450 μ L/ holes, are gently vibrated ELISA Plate, are detected using ELIASA
OD values at 450/630nm wavelength.
(8) TIANZHU XINGNAO Capsul and sample pre-treatments：5g samples are weighed to be placed in clean 50mL centrifuge tubes, respectively add 1,
4th, 16ngAFB1,25mL sample extracting solutions is added, 5min is vibrated after closing the lid, 4000 leave heart 5min, take the μ L of supernatant 50, add
950 μ L sample dilutions, it is to be measured as ELISA sample extracting solutions after mixing.The rate of recovery is added using direct competive ELISA
Experiment, the rate of recovery is respectively 82.4,102.3,95.7%.
(9) configuration of solution：
Carbonic acid buffer：Weigh Na2CO31.59g NaHCO32.93g, addition pure water to 990mL, pH is adjusted to 9.6, then
1000mL is settled to pure water, 4 DEG C of storages are standby.
Phosphate buffer (PBS)：8.5g NaCl, 2.2g Na2HPO4·12H2O, 0.2g NaH2PO4·2H2O, it is dissolved in
In 900mL pure water, pH to 7.4 is adjusted, is settled to 1000mL.
PBST：500mL PBS are taken, add 0.25mL polysorbas20s, are mixed standby.
Nitrite ion A：20mg TMB are dissolved in 10mL DMSO, 4 DEG C of preservations, take out recover to room temperature before use, dilute with pure water
Release to 100mL.
Nitrite ion B：21g monohydrate potassiums, 71.1g Na2HPO4·12H2O, 2g hydrogen peroxide urea, add pure water constant volume
To 1000mL.
Sample extracting solution：4g NaCl are taken, are dissolved in 30mL water, add 70mL methanol, are mixed stand-by.
ELIAS secondary antibody is purchased from SoutherBiotech companies.
Claims (3)
1. hybridoma cell strain AFB1-2A4, it is characterised in that：China General Microbiological culture presevation administrative center has been preserved in,
Deposit number is CGMCC NO.10200, wherein, described hybridoma cell strain is immunized by AFB1-BSA comlete antigen
Mouse simultaneously merges screening acquisition with myeloma.
2. the aspergillus flavus resisting toxin B1 monoclonal antibodies of the hybridoma cell strain AFB1-2A4 secretions described in claim 1, it is special
Sign is：It is produced as secreted by described hybridoma cell strain AFB1-2A4.
3. the application of the aspergillus flavus resisting toxin B1 monoclonal antibodies described in claim 2, it is characterised in that in food security
Aflatoxin B1 immune detection, there is preferable affinity and detection sensitivity to aflatoxin B1.
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