CN107012128A - A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application - Google Patents
A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application Download PDFInfo
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Abstract
The invention provides a kind of hybridoma cell strain F 24 for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, deposit number is CGMCC No.13805.High, the specific good, anti-interference of aspergillus flavus resisting toxin B1 monoclonal antibodies sensitivity of this cell line secretion is good, to 50% inhibition concentration IC of aflatoxin B150For 0.141 μ g/L, cross reacting rate with AFB2 is 9%, cross reacting rate with AFG1 is 19%, with AFG2, AFM1 equal ＜ 1% of cross reacting rate, preferable anti-interference is respectively provided with to auxiliary material and contaminated bacteria for being used in mycotoxin that may be present, agricultural chemicals, heavy metal, concocting process in Chinese medicine etc., immune detection for aflatoxin B1 in Chinese medicine provides condition, with actual application value.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of hybridization of secretion aspergillus flavus resisting toxin B1 monoclonal antibodies
Tumor cell strain and its application.
Background technology
Chinese medicine is possible to because of self property and extraneous factor from processes such as production, harvesting, processing, transport, storages
Influence and contaminant, and then go mouldy and contaminant toxin.The quality of Chinese medicine can not only be influenceed by going mouldy, and lose medicinal material
Original medical value is removed, huge economic loss is caused, and during going mouldy, a variety of mycotoxins of generation can be to suffering from
Liver, kidney, nerve and hemopoietic system of person etc. cause serious infringement, in some instances it may even be possible to induce cancer.Meanwhile, with current traditional Chinese medicine
The popularization of health care's idea, large Chinese medicine of more and more " integration of drinking and medicinal herbs " is used for the daily diet of people and health care is worked as
In, the demand in market is big, if these Chinese medicines are polluted by mycotoxin, will certainly endanger the strong of more crowds
Health, aggravates the financial burden of personal and medical system, more likely further influence society and harmonious stabilization.
Aflatoxin (Aflatoxins, abbreviation AFs) is mainly by aspergillus flavus A.flavus and aspergillus parasiticus
The class that A.parasiticus is produced has the poisonous secondary metabolite of bioactivity, and 17 kinds of AFs phases have been had confirmed at present
The derivative of pass, including aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), and wherein with AFB1
Toxicity it is most strong, therefore 1993 the World Health Organization (WHO) Agency for Research on Cancer (IARC) delimited for I class it is carcinogenic
Thing, and other three kinds of aflatoxin AFB2, AFG1 and AFG2 are successively also divided into 2B class carcinogenic substances.In view of AFs has pole
Strong toxic action, and it is big to the harmfulness of humans and animals, countries in the world and linked groups have formulated harsh limitation mark one after another
It is accurate.《Pharmacopoeia of People's Republic of China》(2015 editions) require to Part of Chinese Medicinal such as polygala, jujube, nutmeg, cassia seed, wheat
Bud, dried orange peel, the fruit of Rangoon creeper, the seed of Oriental arborvitae, the sterculia seed, lotus seeds, peach kernel, betel nut, spina date seed, coix seed etc. must carry out AFB1 detections,
And regulation AFB1 must not exceed 5 μ g/kg.
AFB1 detection method is mainly instrument analytical method, including thin-layered chromatography, high-efficient liquid phase color in current Chinese medicine
Spectrometry, Liquid Chromatography-Mass Spectrometry etc..Thin-layered chromatography is easy to detect, but detection limit for height, it is impossible to quantitative well；Efficiently
The detection such as liquid chromatography, Liquid Chromatography-Mass Spectrometry is accurate, test limit is low, but the complicated sample pretreatment process of needs,
The staff training of special instrument and specialty.These congenital deficiencies of instrument analytical method, make its in actual applications by
Very big limitation is arrived.
Immunoassay can make up all of above shortcoming, and immunoassay is that one kind is combined using antigen and antibody specific
The analysis method of the various materials of reaction detection, the key for setting up the immunoassay method of micromolecular compound is can to produce pair
Micromolecular compound has the antibody of high-affinity and high specific.
The content of the invention
It is an object of the invention to provide a kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies, this is thin
Monoclonal antibody prepared by born of the same parents' strain has preferably specific and higher detection sensitivity to AFB1, to there may be in Chinese medicine
Mycotoxin, agricultural chemicals, heavy metal, the auxiliary material and contaminated bacteria that use in concocting process etc. be respectively provided with preferable anti-interference, be
The immunological detection method for setting up AFB1 in Chinese medicine lays the foundation.
Technical scheme, a kind of hybridoma cell strain of secretion aspergillus flavus resisting toxin B1 monoclonal antibodies, name
For aflatoxin B1 monoclonal antibody hybridoma cell strain F-2-4, Chinese microorganism strain preservation conservator has been preserved in
Meeting common micro-organisms center, deposit number is CGMCC No.13805, and preservation date is on 03 08th, 2017.
Aspergillus flavus resisting toxin B1 monoclonal antibodies, it is the aflatoxin B1 for CGMCC No.13805 by deposit number
Monoclonal antibody hybridoma cell strain F-2-4 secretions are produced, and the aspergillus flavus resisting toxin B1 monoclonal antibodies are applied to Chinese medicine
The analysis detection of middle aflatoxin B1.
The present invention provides the basic preparation process of F-2-4 cell lines：
(1) AFB1 hapten synthesis and identification：100mg aflatoxin B1s and 5mL N are added in 25mL round-bottomed flasks,
Dinethylformamide, mechanical agitation dissolving, is slowly added dropwise 147.3mg POCl3s under the conditions of 0 DEG C, is warming up to 40 DEG C, and
2h is reacted under magnetic agitation, then is warming up to 80 DEG C of continuation stirring reaction 3h, after reaction terminates, natural cooling is poured slowly into frozen water
In, w (NaOH)=10% aqueous solution regulation pH value of solution=7~8 are added, ethyl acetate 30mL is used, extracts three times, merge organic
Phase, anhydrous sodium sulfate drying is evaporated.Absolute ethyl alcohol is recrystallized, and obtains yellow solid aldehyde radical aflatoxin B1 103mg, is
AFB1 haptens, yield 95.37%, proton nmr spectra qualification result；
(2) comlete antigen is synthesized：AFB1 haptens is coupled with bovine serum albumin(BSA) (BSA), ovalbumin (OVA) respectively
Obtain immunizing antigen (AFB1-BSA) and envelope antigen (AFB1-OVA)；
(3) animal immune：6~8 weeks female Balb/c mouse 10 (being divided into two groups of A and B, every group 5) of health are taken, just
Secondary be immunized emulsifies collare dorsal sc multi-point injection with Freund's complete adjuvant, and every mouse immune dosage is 200 μ g AFB1-
BSA；Afterwards booster immunization every two weeks the subcutaneous multi-point injection of nape part once, emulsification incomplete Freund's adjuvant；Last time is exempted from
Epidemic disease replaces incomplete Freund's adjuvant using physiological saline, using intraperitoneal injection, injection dosage and above identical several times；By indirect
ELISA detects serum titer；
(4) cell fusion is screened with cell line：, will be small by polyethylene glycol (PEG4000) method the 3rd day after booster immunization
Mice spleen cell and murine myeloma cell fusion, by complete culture solution culture, are detected using indirect elisa method and secrete AFB1's
Cell line, the inhibition of the cell line is determined using indirect competitive ELISA method, is screened the positive cell strain preferably suppressed and is carried out
Three subclones, it is final to obtain hybridoma cell strain F-2-4；
(5) Antibody preparation purifying and CHARACTERISTICS IDENTIFICATION：Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only；10
Every mouse 0.5mL hybridoma CGMCC No.13805 is expelled to abdominal cavity after it, ascites is collected since the 7th day, by abdomen
Water is purified by caprylic acid-ammonium, and PAGE gel electrophoresis analysis purification effect, the monoclonal antibody of acquisition is imitated
Valency, hypotype, cross reactivity, anti-interference are determined, and are placed in -20 DEG C of preservations；
(6) application of antibody：The hybridoma cell strain F-2-4 aspergillus flavus resisting toxin B1 monoclonal antibodies secreted are used to make
Standby aflatoxin B1 enzyme linked immunological kit, for the analysis detection of aflatoxin B1 in Chinese medicine.
The beneficial effects of the present invention are：
(1) the hybridoma cell strain F-2-4 that the present invention is provided can be used for preparing high-titer aspergillus flavus resisting toxin B1 Dan Ke
Grand antibody, potency >=20000 that aspergillus flavus resisting toxin B1 mouse ascites antibody ELISA methods are measured.
(2) high, the specific good, anti-interference of the aspergillus flavus resisting toxin B1 monoclonal antibodies sensitivity that the present invention is provided is good,
To 50% inhibition concentration IC of aflatoxin B150For 0.141 μ g/L, the cross reacting rate with AFB2 is 9%, the friendship with AFG1
It is 19%, the equal ＜ 1% of cross reacting rate with AFG2, AFM1, to mycotoxin that may be present, agriculture in Chinese medicine to pitch reactivity
Auxiliary material and contaminated bacteria for being used in medicine, heavy metal, concocting process etc. are respectively provided with preferable anti-interference.
(3) the aspergillus flavus resisting toxin B1 monoclonal antibodies that the present invention is provided can be applied to determine aflatoxin B1 in Chinese medicine
Content.
Biological material specimens preservation：The hybridoma cell strain of one plant of secretion aspergillus flavus resisting toxin B1 monoclonal antibody, has been protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, preservation date 2017 year 03 month 08 day, deposit number is CGMCC
No.13805。
Brief description of the drawings
Fig. 1 aflatoxin B1 hapten synthesis route maps
Fig. 2 aflatoxin B1 haptens hydrogen nuclear magnetic resonance spectrograms
Fig. 3 aspergillus flavus resisting toxin B1 monoclonal antibody SDS-PAGE electrophoresis result figures
Fig. 4 aflatoxin B1 competitive ELISA canonical plottings
Fig. 5 aflatoxin B1 competitive ELISA Logit/Log canonical plottings
Embodiment
The following examples of the present invention only further illustrating as present invention, it is impossible in the restriction as the present invention
Perhaps scope.Below by embodiment, the present invention will be further described.
Embodiment 1：Hybridoma cell strain F-2-4 screening
1. hapten synthesis and identification
The synthetic route of AFB1 haptens is as shown in Figure 1.
100mg aflatoxin B1s and 5mL DMFs, mechanical agitation are added in 25mL round-bottomed flasks
Dissolve, 147.3mg POCl3s are slowly added dropwise under the conditions of 0 DEG C, 40 DEG C are warming up to, and react 2h under magnetic stirring, then heat up
To 80 DEG C of continuation stirring reaction 3h, after reaction terminates, natural cooling is poured slowly into frozen water, adds w (NaOH)=10% water
Solution adjusts pH value of solution=7~8, uses ethyl acetate 30mL, extracts three times, merges organic phase, and anhydrous sodium sulfate drying is evaporated.Nothing
Water-ethanol is recrystallized, and obtains yellow solid aldehyde radical aflatoxin B1 103mg, as AFB1 haptens, yield 95.37%.
Take above-mentioned haptens to be identified through proton nmr spectra, as a result see accompanying drawing 2.1H-NMR(CDCl3,300MHz)δ：10.36
(1H, s ,-CHO), 6.78 (1H, s ,-CH-), 6.22 (1H, d, C=C), 4.82 (1H, dd, C=C), 4.31 (1H, dd ,-
CH-), 3.83 (3H, s ,-OCH3), 2.98 (2H, dd, CH2), 2.07 (2H, t, CH2).In collection of illustrative plates, chemical shift δ=10.36
For the aldehyde radical hydrogen resonance absorbing peak of introducing, in addition to the feature hydrogen absworption peak that active compound has in itself, the presence of the hydrogen absworption peak, it was demonstrated that
Spacerarm is coupled successfully, and AFB1 haptens structures are correct.
2. comlete antigen is synthesized
Immunizing antigen is synthesized --- and AFB1 haptens is coupled with BSA
AFB1 haptens 12mg, plus DMF 0.5mL dissolvings are taken, A liquid is obtained；50mg BSA separately are taken, plus
PH5.4 0.05mol/L acetate buffers dissolve, and obtain B liquid；A liquid is added drop-wise in B liquid dropwise, 4 DEG C are stirred overnight, plus
5mg sodium borohydrides, continue to stir 2h, 0.05mol/L phosphate buffers are dialysed three days, liquid are changed daily 3 times, are encapsulated, -20 DEG C of guarantors
Deposit standby.
Envelope antigen is synthesized --- and AFB1 haptens is coupled with OVA
AFB1 haptens 6mg, plus dimethyl sulfoxide (DMSO) 0.5mL dissolvings are taken, A liquid is obtained；50mg OVA separately are taken, plus
PH5.40.05mol/L acetate buffers dissolve, and obtain B liquid；A liquid is added drop-wise in B liquid dropwise, 4 DEG C are stirred overnight, plus 3mg
Sodium borohydride, continues to stir 2h, 0.05mol/L phosphate buffers are dialysed three days, liquid are changed daily 3 times, are encapsulated, -20 DEG C of preservations
It is standby.
3. animal immune
Take 6~8 weeks female Balb/c mouse 10 (being divided into two groups of A and B, every group 5) of health, initial immunity Freund
Freund's complete adjuvant emulsifies collare dorsal sc multi-point injection, and every mouse immune dosage is 200 μ g AFB1-BSA；Strengthen exempting from afterwards
Epidemic disease every two weeks the subcutaneous multi-point injection of nape part once, emulsification incomplete Freund's adjuvant；Last time is immune to use physiological saline
Instead of incomplete Freund's adjuvant, using intraperitoneal injection, injection dosage and above identical several times.Specifically immune step is shown in Table 1.
The mouse immune program of table 1
For the third time, four times, 7d after booster immunization, blood is taken to mouse docking, and ELISA method determines mice serum potency, tool
Body step is as follows：
(1) envelope antigen (AFB1-OVA) is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, often
The μ L coated elisa plates of hole 100,37 DEG C of incubation 2h, are got rid of coating buffer, are washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid are patted dry；
(3) 50 μ L are added per hole with the antiserum of PBS doubling dilutions, 25 DEG C of reaction 30min hypsokinesis dereaction liquid, with PBST
Washing 3~5 times, per minor tick 30s, is patted dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines OD value of the wavelength in 450nm, with sample well OD450Extension rate close to 1 is used as the positive
The potency of serum.
4. cell fusion
(1) prepared by feeder cells：Disconnected neck puts to death 8~10 week old Balb/c mouse, is immersed in 5min in 75% alcohol, immediately
It is put into superclean bench, belly is put in plate or be fixed on dissection plate upward.Mouse web portion skin is played with ophthalmic tweezers sub-folder
Skin, an osculum is cut with scissors, notes being sure not to break peritonaeum, in order to avoid peritoneal fluid outflow and pollution.Then scissors is used to both sides up and down
Blunt separation is done, peritonaeum is fully exposed.Peritonaeum is wiped with cotton ball soaked in alcohol to sterilize.5mL RPMI-1640 bases are drawn with syringe
Nutrient solution, injects mouse peritoneal, gently draws back syringe, rock mouse leg and afterbody several times.Abdominal cavity is drawn back with original annotation emitter
Interior liquid, injects centrifuge tube.So operate 3~4 times repeatedly.1000r/min centrifuges 10min, abandons supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and 100 μ L/ holes are added drop-wise to culture plate, put incubator standby.
(2) prepared by splenocyte：3d after booster immunization, takes immune Balb/c mouse, dislocates and put to death after eye socket blood sampling, 75%
Spleen is taken after being sterilized in alcohol, connective tissue is removed, prepares splenocyte suspension, be transferred in 50mL centrifuge tubes, plus RPMI-1640
To 30mL, 1500~2000r/min centrifugation 5min, supernatant, plus RPMI-1640 to 30mL are abandoned, is counted stand-by.
(3) prepared by myeloma cell：(the viable count for taking 3 bottles of growth conditions good>95%) myeloma cell, by it is complete
Full blow down, be transferred in 50mL centrifuge tubes, plus RPMI-1640 to 30mL, 1500~2000r/min centrifugation 5min, supernatant is abandoned, plus
RPMI-1640 to 30mL, is counted stand-by.
(4) mixing with cells：Splenocyte:Myeloma cell=8:1, mixing, 1500~2000r/min centrifugations 5min.
(5) cell fusion：By the cell mixed centrifugation, dry supernatant, sedimentation cell block bullet into pasty state, puts 37 DEG C of water
Bath, adds 1mL fusion agents in 1min, and fusion agent is polyethylene glycol (PEG) 4000, acts on 2min, and is gently mixed cell,
The PEG nutrient solutions of 20mL serum-frees are added in subsequent 4min, 1000r/min centrifugation 10min abandon supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and cover plant, per the μ L of hole 100, is put in incubator in 96 porocyte culture plates containing feeder cells.
5. cell line is screened
When the 1/2~1/3 of cell length to bottom hole, you can carry out antibody test.Using ELISA method to there is hybridoma thin
The culture hole of intracellular growth is screened, and screening is in two steps：The first step first filters out positive cell hole, second step with indirect ELISA
It is standard items from AFB1, AFB2, AFG1, AFG2, AFM1, inhibition survey is carried out to positive cell with indirect competitive ELISA
It is fixed.Selecting has preferable suppression to AFB1 standard items, and the hole not suppressed to AFB2, AFG1, AFG2, AFM1 standard items is adopted
It is subcloned, is detected with same method with limiting dilution assay.In triplicate, you can obtaining can stably excreting aspergillus flavus-resistance
The cell line F-2-4 of mould toxin B1 monoclonal antibodies.The cell line was preserved in China Microbiological bacterium on 03 08th, 2017
Plant preservation administration committee common micro-organisms center (abbreviation CGMCC, address：Datun Road, Chaoyang District, Beijing City, the Chinese Academy of Sciences is micro-
Biological study institute, postcode 100101), deposit number is CGMCC No.13805.
Embodiment 2：The preparation purifying of aspergillus flavus resisting toxin B1 monoclonal antibodies and CHARACTERISTICS IDENTIFICATION
1. it is prepared by ascites
Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only.By the hybridization in exponential phase after 10 days
Oncocyte CGMCC No.13805 are collected with RPMI-1640 basal mediums, and with blood counting chamber and microscopic counting, cell is dense
Degree is 1.0 × 106~1.5 × 106In the range of individual/mL.Every mouse 0.5mL hybridoma CGMCC No.13805 is expelled to
Abdominal cavity.Notice that observation mouse web portion after one week expands, ascites is gathered in mouse peritoneal with asepsis injector, every one to two days
Collection once, is so repeatedly gathered until mouse natural death repeatedly.5000r/min centrifuges 5min at 4 DEG C, collects supernatant, and
Remove the fat and albuminous membranae of ascites upper strata floating.
2. antibody purification
Monoclonal antibody is comprised the following steps that using octanoic acid-ammonium sulfate method purifying：
(1) ascites is taken out into thaw at RT from -20 DEG C of refrigerators.Ascites is filtered with double-layer filter paper, it is preliminary remove fatty piece, it is thin
Born of the same parents' fragment and other impurities.12000r/min centrifuges 15min, takes supernatant, abandons precipitation.Precise volume ascites volume；
The acetate buffer magnetic agitation of the ascites of (2) 1 parts of volumes and 3 parts of volumes is mixed, and pH is adjusted with 2mol/L HCl
To 4.5~4.8；
(3) caprylic acid is slowly added under magnetic agitation, 1mL ascites adds 33 μ L caprylic acids, adds rear room temperature magnetic agitation
30min, rearmounted 4 DEG C of standings 2h；
(4) 12000r/min centrifuges 5min, takes supernatant, and filtrate is collected in double-layer filter paper filtering；
(5) filtrate volume is measured, the 0.1mol/L pH7.4 of 1/10 volume PBS is added, with 2mol/L NaOH (records
NaOH volumes) adjust pH to 7.4；
(6) by supernatant ice bath precooling, plus ammonium sulfate solids are to 0.277g/mL, stirring while adding, and in being added in 30min,
Put 4 DEG C overnight；
(7) 12000r/min centrifuges 15min, abandons supernatant.Precipitation is dissolved with the 0.01mol/L PBS of certain volume.Use PB
Dialysis changes 0.01mol/L PBSs two days two days later, collects dialyzate, and 12000r/min centrifugation 15min take supernatant, put -20
DEG C preserve；
(8) antibody of purifying analyzes purification effect with PAGE gel electrophoresis, as a result sees accompanying drawing 3, antibody is in denaturation
After SDS-PAGE electrophoresis, destination protein band size is with being expected unanimously.Antibody can be dissociated into two subunits, large subunit (heavy chain,
50kD) with a small subunit (light chain, 25kD), illustrate that purity is good.
3. antibody titer is determined
Determined, concretely comprised the following steps using indirect ELISA method：
(1) envelope antigen (AFB1-OVA) is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000、1:
2000、1:4000、1:8000 dilutions, per the μ L coated elisa plates of hole 100,37 DEG C of incubation 2h get rid of coating buffer, 1 are washed with PBST
It is secondary, pat dry；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid are patted dry；
(3) monoclonal antibody is done 1 with PBS:5000、1:10000、1:20000、1:40000、1:80000 gradient dilutions,
ELIAS secondary antibody is diluted 1000 times simultaneously, the antibody diluted does 10 with ELIAS secondary antibody:1 mixing, 100 μ L are added per hole, 25 are put
45min is incubated in DEG C incubator, each concentration does a repetition, is washed 3~5 times with PBST, per minor tick 30s, patted dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines wavelength in 450nm OD values, the results are shown in Table 2.
The titration result of table 2
As shown in Table 2, detect, using antibody concentration of the OD values more than 1.0 as potency, be as a result shown in anti-with chessboard method
Original is coated with 1 respectively:1000、1:2000、1:4000、1:When 8000, the potency of 5 batches of antibody is respectively 1:80000、1:80000、1:
40000、1:20000 (the 1st batches), 1:80000、1:80000、1:40000、1:20000 (the 2nd batches), 1:80000、1:80000、
1:40000、1:40000 (the 3rd batches), 1:80000、1:80000、1:40000、1:40000 (the 4th batches), 1:40000、1:
40000、1:40000、1:20000 (the 5th batches), it is seen then that potency >=20000 of aspergillus flavus resisting toxin B1 monoclonal antibodies.
4. antibody subtype is identified
Identify that the aspergillus flavus resisting toxin B1 of hybridoma cell strain F-2-4 secretions is mono- with commercially available SIGMA hypotypes identification kit
The hypotype of clonal antibody is IgG1Type.
5. antibody cross reaction is determined
Determined, concretely comprised the following steps using indirect competitive ELISA method：
(1) envelope antigen (AFB1-OVA) is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, often
The μ L coated elisa plates of hole 100,37 DEG C of incubation 2h, are got rid of coating buffer, are washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid are patted dry；
(3) first added per hole aflatoxin standard working solution that 50 μ L are serially diluted (concentration is respectively 0,0.05,
0.15th, 0.45,1.35 μ g/L), then add 50 μ L and do 1 with PBS:The monoclonal antibody of 20000 dilutions, 25 DEG C of reaction 30min
Hypsokinesis dereaction liquid, is washed 3~5 times with PBST, per minor tick 30s, is patted dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines OD value of the wavelength in 450nm.
Using the logarithm value of aflatoxin concentration as abscissa, with percentage absorbance (each concentration standards OD values with not
The percentage of the quasi- sample wells OD values of mark-on) draw standard curve for ordinate.It is dense with the quality of each percentage absorbance of curve 50%
Spend (IC50) cross reacting rate is calculated as follows, 3 are the results are shown in Table, cross reacting rate is smaller, antibody specificity is higher.
In formula：
Si--- cross reacting rate, %；
Y --- the IC of AFB1 standard working solution curves50Value；
Z --- the IC of aflatoxin standard working solution curve beyond AFB150Value.
The cross reaction measurement result of table 3
As shown in Table 3, aspergillus flavus resisting toxin B1 monoclonal antibodies are smaller to other kinds of aflatoxin cross reaction,
It is specific preferable.
6. antibody anti-interference is determined
The standard curve of aflatoxin B1 is drawn according to the determination step of 5. cross reactivities, while respectively will be following dry
Disturbing material add value listed by form is diluted to standard items buffer solution is used to analyze, and is developed the color corresponding OD values, tied according to medicine
The alluvial that standardization tracing analysis goes out calculates cross reacting rate (cross reacting rate=alluvial/add value × 100%), as a result
It is shown in Table 4.
Anti-interference data of the table 4 to other materials common in Chinese medicine
As shown in Table 4, the auxiliary material that is used in Chinese medicine in mycotoxin that may be present, agricultural chemicals, heavy metal, concocting process and
The property of contaminated bacteria confrontation aflatoxin B1 monoclonal antibody is not interfered with.
Embodiment 3：The application of aspergillus flavus resisting toxin B1 monoclonal antibodies
The hybridoma cell strain F-2-4 aspergillus flavus resisting toxin B1 monoclonal antibodies secreted are used to prepare aflatoxin B1
Enzyme linked immunological kit, for the analysis detection of aflatoxin B1 in Chinese medicine.
1. the composition of enzyme linked immunological kit
(1) it is coated with the ELISA Plate of aspergillus flavus resisting toxin B1 monoclonal antibodies；
(2) enzyme mark aflatoxin B1 antigen：Envelope antigen and horseradish peroxidase (HRP) described in embodiment 1
Coupling is carried out to prepare, and with enzyme-labelled antigen diluted to best effort concentration；
(3) aflatoxin B1 standard items：Standard solution concentration be respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L,
0.45μg/L、1.35μg/L；
(4) substrate nitrite ion：It is made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, B liquid is 1% tetramethyl
The aqueous solution of benzidine；
(5) terminate liquid：2mol/L sulfuric acid solutions；
(6) cleaning solution：Prepared as follows per 1L cleaning solutions：By 10mL Tween-20s, 5g sodium azide and 990mL
Phosphate buffer is mixed；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.4；
(7) liquid is redissolved：Liquid is redissolved per 1L to prepare as follows：12g caseins phosphate buffer is dissolved simultaneously
It is settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.4.
2. the preparation of reagent constituents
(1) preparation of the ELISA Plate of coating aspergillus flavus resisting toxin B1 monoclonal antibodies
Aspergillus flavus resisting toxin B1 monoclonal antibodies are diluted to best effort concentration with coating buffer solution, 96 hole polyphenyl are coated with
Ethene ELISA Plate, per hole 100 μ L, 37 DEG C of incubation 2h, gets rid of coating buffer, is washed with cleaning solution 1 time, each 30s is patted dry, then
Liquid in 150 μ L confining liquids, 37 DEG C of incubation 2h, hole of inclining is added in every hole, is preserved after drying with aluminium film vacuum sealing.
It is coated with buffer solution：PH9.6,0.05mol/L carbonate buffer solution；
Confining liquid：Prepared as follows per 1L confining liquids：5mL horse serums, 1g sodium azide, 30g caseins are mixed
Close, dissolved with phosphate buffer and be settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is
7.2。
(2) preparation of enzyme mark aflatoxin B1 antigen
Envelope antigen 15mg described in Example 1, adds the dissolving of 300 μ L DMFs, adds carbonization
Diimine 10mg and n-hydroxysuccinimide 10mg, priming reaction 0.5h；With 0.05mol/L, pH9.0 CB buffer solutions
0.5mL, dissolves horseradish peroxidase 20mg, adds 2mL water, then adds the above-mentioned μ L of activating solution 300, adds 1mol/L hydrogen-oxygens
Change the μ L of sodium 30, react at room temperature 4h；With 0.02mol/L PBSs 2 days, liquid is changed daily 4 times, taking-up obtains dialyzate 2.8mL；Plus
Enter BSA protected proteins, 5/10000ths preservatives, and with enzyme-labelled antigen diluted to best effort concentration, dispense and preserve.
Enzyme-labelled antigen dilution：Prepared as follows per 1L enzyme-labelled antigen dilutions：8g bovine serum albumin(BSA)s are used
Phosphate buffer dissolves and is settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.2.
3. kit test method
(1) sample pre-treatments
The Chinese medicine sample of 1.0g crushing is weighed into 50mL centrifuge tubes, 10mL methanol is added, vibrates 5min, 3000g room temperatures
(20-25 DEG C/68-77 ℉) centrifuges 5min；Take in the clean centrifuge tubes of 2mL to 10mL, in 20-30 DEG C of (68-86 ℉) water-bath nitrogen
Flow down drying；Add 2mL deionized water whirling motion 1min, add 6mL chloroforms vibration 5min, 3000g room temperatures (20-25 DEG C/
68-77 ℉) centrifugation 5min；Remove upper strata aqueous phase, remove layer 3mL in drying under 20-30 DEG C of (68-86 ℉) water-bath nitrogen stream；Plus
Enter 1mL n-hexane whirling motion 2min, add 1mL and redissolve liquid whirling motion 3min, 3000g room temperature (20-25 DEG C/68-77 ℉) centrifugation
5min；Remove upper organic phase, remove layer and analyzed.
(2) detected with kit
AFB1 standard solutions/μ L/ holes of sample liquid 50 are added into plate hole, the μ L/ holes of enzyme mark AFB1 antigens 50 gently vibrate
Mix, react 45min with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate, pour out liquid in hole, the μ of cleaning solution 250 is added per hole
Liquid in hole is poured out after L, 30s, common board-washing is repeated operation 5 times, is patted dry with blotting paper；Substrate nitrite ion A liquid is added per hole
With each 50 μ L of B liquid, gently vibration is mixed, and 15min is reacted with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate；Add and terminate per hole
The μ L of liquid 50, gently vibration is mixed, and setting ELIASA determines the absorbance per hole at 450nm.
(3) result is calculated
Calculate percentage absorbance：With the standard solution or sample liquid absorbance obtained and blank solution absorbance
The ratio of value is calculated, and sees below formula：
In formula：W --- percentage absorbance, %；
The mean absorbance values of A --- standard solution or sample liquid；
A0--- the mean absorbance values of blank solution (concentration is 0 μ g/L standard solution).
Draw standard curve：With the percentage absorbance (%) of calculating for ordinate, the logarithm value of standard solution concentration
(log10) standard curve is drawn for abscissa.
Sample results are calculated：The percentage absorbance of sample liquid is substituted into standard curve, sample is read from standard curve
After concentration corresponding to this, the content of aflatoxin B1 is calculated as follows in sample：
X=C × n
X=C × n
In formula：X --- the content of aflatoxin B1 in sample, μ g/kg；
C --- the concentration of aflatoxin B1 from the sample checked on standard curve, μ g/L；
N --- Sample Dilution multiple.
It can also be calculated with the data processing software of various ELIASAs.
Note：Result of calculation deducts blank value, and acquired results retain a decimal.
4. Detection results evaluation
(1) linear relationship
Concentration of standard solution is respectively 0,0.05,0.15,0.45,1.35 μ g/L, and the OD values of measure are shown in Table 5.Inhaled with percentage
Shading value (W, %) is ordinate, and the logarithm value (log10) of concentration of standard solution draws standard curve (see accompanying drawing for abscissa
4).Using logitW as ordinate, the logarithm value of concentration of standard solution is abscissa, is understood after accompanying drawing 4 is changed, in 0.05 μ g/L
In~1.35 μ g/L concentration ranges, good linear relationship (see accompanying drawing 5) is presented with aflatoxin B1 log concentration in logitW,
Obtain regression equation Y=-2.393X-2.327, R2=0.994.
The OD value measurement results of the standard liquid of table 5
(2) method test limit and quantitative limit
Reference《Pharmacopoeia of People's Republic of China》The drug standard analysis method verification guide principle of version 9101 in 2015,
δ in method test limit LOD=3.3 δ/S, quantitative limit LOQ=10 δ/S, formula：Determine the standard deviation of blank value；S：Standard curve
Slope.
Understood according to above-mentioned (1), δ=0.023, S=2.393, it is thus determined that test limit LOD is 0.032 μ g/L, quantitative limit
LOQ is 0.096 μ g/L.
(3) pattern detection is limited
Using the blank malt of separate sources, peach kernel, spina date seed, the sterculia seed, stiff silkworm, centipede, coix seed, scorpio, leech,
Lotus seeds, the fruit of Rangoon creeper, dried orange peel, polygala, cassia seed, jujube, betel nut, earthworm, each 20 parts of seed of Oriental arborvitae sample are measured, test limit with
Determine average value and add 3 times of standard deviation determinations, the results are shown in Table 6.
The pattern detection of table 6 limits result
| Sample | Test limit/(ng/kg) | Sample | Test limit/(ng/kg) | Sample | Test limit/(ng/kg) |
| Malt | 285.7 | Peach kernel | 592.9 | Spina date seed | 437.2 |
| The sterculia seed | 382.3 | Stiff silkworm | 217.5 | Centipede | 339.8 |
| Coix seed | 159.9 | Scorpio | 388.6 | Leech | 174.7 |
| Lotus seeds | 383.5 | The fruit of Rangoon creeper | 291.5 | Dried orange peel | 235.5 |
| Polygala | 267.8 | Cassia seed | 371.0 | Jujube | 397.0 |
| Betel nut | 420.3 | Earthworm | 337.1 | The seed of Oriental arborvitae | 355.8 |
(4) degree of accuracy (rate of recovery) and precision (repeatability)
With the malt without AFB1, peach kernel, spina date seed, the sterculia seed, stiff silkworm, centipede, coix seed, scorpio, leech, lotus seeds,
The fruit of Rangoon creeper, dried orange peel, polygala, cassia seed, jujube, betel nut, earthworm, the seed of Oriental arborvitae are blank sample matrix, carry out three concentration levels
Addition recovery test, respectively with three batch kit measurements, calculate sample TIANZHU XINGNAO Capsul and batch in, interassay coefficient of variation,
It the results are shown in Table 7.
The sample veracity and precision result of table 7
As a result show, malt, peach kernel, spina date seed, the sterculia seed, stiff silkworm, centipede, coix seed, scorpio, leech, lotus seeds, make monarch
Son, dried orange peel, polygala, cassia seed, jujube, betel nut, earthworm, the TIANZHU XINGNAO Capsul of 18 kinds of matrix of the seed of Oriental arborvitae are criticized 60%~120%
The equal ＜ 15% of interior, interassay coefficient of variation.
(4) kit storage life
Kit preservation condition was 2-8 DEG C, by the measure of 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, AFB1 TIANZHU XINGNAO Capsuls are in normal range (NR).In view of having improper guarantor during transport and use
Condition appearance is deposited, kit is placed 8 days at 37 DEG C and carries out accelerated aging tests, as a result the indices of the kit are complete
Meet the requirements；Situation is freezed in view of kit, kit is placed 8 days in -20 DEG C of refrigerators, as a result the kit
Indices are also completely normal.It can show that kit can at least be preserved more than 12 months at 2-8 DEG C from result above.
Claims (3)
1. a kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies, it is characterised in that：It is named as aspergillus flavus
Toxin B1 monoclonal antibody hybridoma cells strain F-2-4, has been preserved in China Committee for Culture Collection of Microorganisms commonly micro-
Bio-Centers, deposit number is CGMCC No.13805, and preservation date is on 03 08th, 2017.
2. a kind of aspergillus flavus resisting toxin B1 monoclonal antibodies, it is characterised in that：It is to be as deposit number described in claim 1
What CGMCC No.13805 aflatoxin B1 monoclonal antibody hybridoma cell strain F-2-4 secretions were produced.
3. the application of aspergillus flavus resisting toxin B1 monoclonal antibodies described in claim 2, it is characterised in that：For aspergillus flavus in Chinese medicine
Toxin B1 analysis detection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112881697A (en) * | 2021-01-13 | 2021-06-01 | 北京中检葆泰生物技术有限公司 | Method for stably detecting aflatoxin content |
| CN113583135A (en) * | 2020-01-17 | 2021-11-02 | 中国医学科学院药用植物研究所 | High-sensitivity anti-aflatoxin B1 monoclonal antibody and application thereof |
| CN113712967A (en) * | 2021-09-17 | 2021-11-30 | 华中农业大学 | Application of compound in preparation of anti-aflatoxin B1 toxicity medicine |
| CN115341010A (en) * | 2021-05-13 | 2022-11-15 | 华中农业大学 | Target participating in aflatoxin B1 toxic effect and application thereof |
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| CN102747043A (en) * | 2012-04-20 | 2012-10-24 | 中国农业科学院油料作物研究所 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
| CN104762267A (en) * | 2015-01-15 | 2015-07-08 | 北京科技大学 | Hybridoma cell AFB1-2A4 and aflatoxin B1 monoclonal antibody produced by hybridoma cell AFB1-2A4 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102747043A (en) * | 2012-04-20 | 2012-10-24 | 中国农业科学院油料作物研究所 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
| CN104762267A (en) * | 2015-01-15 | 2015-07-08 | 北京科技大学 | Hybridoma cell AFB1-2A4 and aflatoxin B1 monoclonal antibody produced by hybridoma cell AFB1-2A4 |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113583135A (en) * | 2020-01-17 | 2021-11-02 | 中国医学科学院药用植物研究所 | High-sensitivity anti-aflatoxin B1 monoclonal antibody and application thereof |
| CN113583135B (en) * | 2020-01-17 | 2023-06-23 | 中国医学科学院药用植物研究所 | High-sensitivity anti-aflatoxin B1 monoclonal antibody and application thereof |
| CN112881697A (en) * | 2021-01-13 | 2021-06-01 | 北京中检葆泰生物技术有限公司 | Method for stably detecting aflatoxin content |
| CN115341010A (en) * | 2021-05-13 | 2022-11-15 | 华中农业大学 | Target participating in aflatoxin B1 toxic effect and application thereof |
| CN113712967A (en) * | 2021-09-17 | 2021-11-30 | 华中农业大学 | Application of compound in preparation of anti-aflatoxin B1 toxicity medicine |
| CN113712967B (en) * | 2021-09-17 | 2023-09-22 | 华中农业大学 | Application of compound in preparation of anti-aflatoxin B1 toxicity drugs |
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