CN107083368A - A kind of hybridoma cell strain for secreting anti-total aflatoxina monoclonal antibody and its application - Google Patents
A kind of hybridoma cell strain for secreting anti-total aflatoxina monoclonal antibody and its application Download PDFInfo
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Abstract
The invention provides a kind of hybridoma cell strain F 31 for secreting anti-total aflatoxina monoclonal antibody, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, deposit number is CGMCC No.13806.High, the specific good, anti-interference of anti-total aflatoxina monoclonal antibody sensitivity of this cell line secretion is good, to aflatoxin B150% inhibition concentration IC50For 0.046 μ g/L, with AFB2Cross reacting rate be 110%, with AFG1Cross reacting rate be 96%, with AFG2Cross reacting rate be 78%, 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., is aflatoxin B in Chinese medicine1、B2、G1、G2The immune detection of total amount 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 the anti-total aflatoxina monoclonal antibody of secretion
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 AFB1Poison
Property it is most strong, therefore 1993 the World Health Organization (WHO) Agency for Research on Cancer (IARC) delimited as I class carcinogenic substance, and
Other three kinds of aflatoxin AFB2、AFG1And AFG2Successively it is also divided into 2B class carcinogenic substances.In view of AFs has extremely strong poison
Property effect, and big to the harmfulness of humans and animals, countries in the world and linked groups have formulated harsh limit standard one after another.《In
Magnificent people's republic's pharmacopeia》(2015 editions) require to Part of Chinese Medicinal for example polygala, jujube, nutmeg, cassia seed, malt, 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 aflatoxin detection, and
Provide AFG2、AFG1、AFB2、AFB1Total amount must not exceed 10 μ g/kg.
The detection method of aflatoxin is mainly instrument analytical method in current Chinese medicine, including thin-layered chromatography, efficiently
Liquid chromatography, Liquid Chromatography-Mass Spectrometry etc..Thin-layered chromatography is easy to detect, but detection limit for height, it is impossible to fixed well
Amount；The detection such as high performance liquid chromatography, Liquid Chromatography-Mass Spectrometry is accurate, test limit is low, but needs to locate before complicated sample
The staff training of reason process, special instrument and specialty.These congenital deficiencies of instrument analytical method, make it actually should
It is limited by very large in.
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 anti-total aflatoxina monoclonal antibody, this is thin
Monoclonal antibody prepared by born of the same parents' strain is to AFB1、AFB2、AFG1、AFG2Preferably specific and higher detection sensitivity is respectively provided with,
Auxiliary material and contaminated bacteria for being used in mycotoxin that may be present, agricultural chemicals, heavy metal, concocting process in Chinese medicine etc. are respectively provided with compared with
Good anti-interference, lays the foundation for the immunological detection method of setting up total aflatoxin content in Chinese medicine.
Technical scheme, a kind of hybridoma cell strain of the anti-total aflatoxina monoclonal antibody of secretion, name
For anti-total aflatoxina monoclonal antibody hybridoma cell strain F-3-1, Chinese microorganism strain preservation management committee has been preserved in
Member's meeting common micro-organisms center, deposit number is CGMCC No.13806, and preservation date is on 03 08th, 2017.
Anti- total aflatoxina monoclonal antibody, it is anti-total aspergillus flavus poison for CGMCC No.13806 by deposit number
What plain monoclonal antibody hybridoma cell strain F-3-1 secretions were produced, during the anti-total aflatoxina monoclonal antibody is applied to
Aflatoxin B in medicine1、B2、G1、G2The analysis detection of total amount.
The present invention provides the basic preparation process of F-3-1 cell lines：
(1) AFs hapten synthesis and identification：100mg aflatoxin Bs are added in 25mL round-bottomed flasks1With 5mL N,
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 B1103mg, as AFs
Haptens, yield 95.37%, proton nmr spectra qualification result；
(2) comlete antigen is synthesized：AFs haptens is coupled with bovine serum albumin(BSA) (BSA), ovalbumin (OVA) respectively
Obtain immunizing antigen (AFs-BSA) and envelope antigen (AFs-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 AFs-BSA；
Afterwards booster immunization every two weeks the subcutaneous multi-point injection of nape part once, emulsification incomplete Freund's adjuvant；Last time is immune to be made
Incomplete Freund's adjuvant is replaced with 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, utilize indirect elisa method detection secretion AFB1's
Cell line, the inhibition of the cell line is determined using indirect competitive ELISA method, is screened to AFB1、AFB2、AFG1、AFG2Have
The positive cell strain of suppression carries out three subclones, final to obtain hybridoma cell strain F-3-1；
(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.13806 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-3-1 anti-total aflatoxina monoclonal antibodies secreted are used to make
Standby aflatoxin enzyme linked immunological kit, for aflatoxin B in Chinese medicine1、B2、G1、G2The analysis detection of total amount.
The beneficial effects of the present invention are：
(1) the hybridoma cell strain F-3-1 that the present invention is provided can be used for preparing the anti-total aflatoxina Dan Ke of high-titer
Grand antibody, potency >=9 × 10 that anti-total aflatoxina mouse ascites antibody ELISA method is measured6。
(2) high, the specific good, anti-interference of the anti-total aflatoxina monoclonal antibody sensitivity that the present invention is provided is good,
To aflatoxin B150% inhibition concentration IC50For 0.046 μ g/L, with AFB2Cross reacting rate be 110%, with AFG1's
Cross reacting rate is 96%, with AFG2Cross reacting rate be 78%, to mycotoxin that may be present in Chinese medicine, agricultural chemicals, again
Auxiliary material and contaminated bacteria for being used in metal, concocting process etc. are respectively provided with preferable anti-interference.
(3) the anti-total aflatoxina monoclonal antibody that the present invention is provided can be applied to determine aflatoxin in Chinese medicine
B1、B2、G1、G2Total amount.
Biological material specimens preservation：The hybridoma cell strain of one plant of anti-total aflatoxina monoclonal antibody of secretion, 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.13806。
Brief description of the drawings
Fig. 1 aflatoxin hapten synthesis route maps
Fig. 2 aflatoxin haptens hydrogen nuclear magnetic resonance spectrograms
The anti-total aflatoxina monoclonal antibody SDS-PAGE electrophoresis result figures of Fig. 3
Fig. 4 aflatoxin competitive ELISA canonical plottings
Fig. 5 aflatoxin 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-3-1 screening
1. hapten synthesis and identification
The synthetic route of AFs haptens is as shown in Figure 1.
100mg aflatoxin Bs are added in 25mL round-bottomed flasks1With 5mL DMFs, mechanical agitation
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 B1103mg, as AFs 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 AFs haptens structures are correct.
2. comlete antigen is synthesized
Immunizing antigen is synthesized --- and AFs haptens is coupled with BSA
AFs 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 AFs haptens is coupled with OVA
AFs 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 AFs-BSA；Booster immunization afterwards
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 generation
For 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 (AFs-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
From AFB1、AFB2、AFG1、AFG2For standard items, inhibition measure is carried out to positive cell with indirect competitive ELISA.Select
To AFB1、AFB2、AFG1、AFG2Standard items are respectively provided with the hole preferably suppressed, are subcloned using limiting dilution assay, with same
Method detected.In triplicate, you can obtain the cell line F- of the energy anti-total aflatoxina monoclonal antibody of stably excreting
3-1.The cell line was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 03 08th, 2017
The heart (abbreviation CGMCC, address：Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preservation
Numbering is CGMCC No.13806.
Embodiment 2：The preparation purifying of anti-total aflatoxina monoclonal antibody 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.13806 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.13806 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,
45kD) 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 (AFs-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 are got rid of coating buffer, washed 1 time with PBST, clapped
It is 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:105、1:3×106、1:9×106、1:2.7×107、1:8.1×107Gradient
Dilution, while ELIAS secondary antibody is diluted into 1000 times, the antibody diluted does 10 with ELIAS secondary antibody:1 mixing, 100 μ L are added per hole,
Put and 45min is incubated in 25 DEG C of incubators, each concentration does a repetition, 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 1:1:2.7×107、1:1:
2.7×107、1:9×106、1:9×106(the 1st batch), 1:1:2.7×107、1:1:2.7×107、1:9×106、1:9×106
(the 2nd batch), 1:1:2.7×107、1:1:2.7×107、1:9×106、1:9×106(the 3rd batch), 1:1:2.7×107、1:1:2.7
×107、1:9×106、1:9×106(the 4th batch), 1:1:2.7×107、1:1:2.7×107、1:9×106、1:9×106(the 5th
Batch), it is seen then that potency >=9 × 10 of anti-total aflatoxina monoclonal antibody6。
4. antibody subtype is identified
The anti-total aflatoxina list of hybridoma cell strain F-3-1 secretions is identified 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 (AFs-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——AFB1The IC of standard working solution curve50Value；
z——AFB1The IC of aflatoxin standard working solution curve in addition50Value.
The cross reaction measurement result of table 3
As shown in Table 3, anti-total aflatoxina monoclonal antibody has cross reaction to four kinds of aflatoxin, illustrates this
Antibody can be used for determining AFB1、AFB2、AFG1、AFG2Total amount.
6. antibody anti-interference is determined
Aflatoxin B is drawn according to the determination step of 5. cross reactivities1Standard curve, 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 total aflatoxina monoclonal antibody is not interfered with.
Embodiment 3：The application of anti-total aflatoxina monoclonal antibody
The hybridoma cell strain F-3-1 anti-total aflatoxina monoclonal antibodies secreted are used to prepare aflatoxin enzyme
Linked immunoassay reagent kit, for aflatoxin B in Chinese medicine1、B2、G1、G2The analysis detection of total amount.
1. the composition of enzyme linked immunological kit
(1) it is coated with the ELISA Plate of anti-total aflatoxina monoclonal antibody；
(2) enzyme mark aflatoxin antigen：Envelope antigen described in embodiment 1 enters with horseradish peroxidase (HRP)
Row coupling is prepared, and with enzyme-labelled antigen diluted to best effort concentration；
(3) aflatoxin B1Standard items：Standard solution concentration is respectively 0 μ g/L, 0.03 μ g/L, 0.1 μ g/L, 0.2 μ
g/L、0.6μg/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) it is coated with the preparation of the ELISA Plate of anti-total aflatoxina monoclonal antibody
Anti- total aflatoxina monoclonal antibody is 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 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
AFB is added into plate hole1The μ L/ holes of standard solution/sample liquid 20, the μ L/ holes of enzyme mark aflatoxin antigen 80, gently
Light vibration is mixed, and is reacted 45min with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate, is poured out liquid in hole, and washing is added per hole
Liquid in hole is poured out after liquid 250 μ L, 30s, common board-washing is repeated operation 5 times, is patted dry with blotting paper；Substrate colour developing is added per hole
Liquid A liquid and each 50 μ L of B liquid, gently vibration are mixed, and 15min is reacted with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate；Add per hole
Enter the μ L of terminate liquid 50, gently vibration is mixed, 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, aflatoxin B in sample1、B2、G1、G2Total amount be calculated as follows：
X=C × n
In formula：X --- the total amount of aflatoxin in sample, μ g/kg；
C --- the concentration of aflatoxin 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 0,0.03,0.1,0.2,0.6 μ g/L, and the OD values of measure are shown in Table 5.With percentage absorbance
It is ordinate to be worth (W, %), and the logarithm value (log10) of concentration of standard solution draws standard curve for abscissa (see accompanying drawing 4).With
LogitW is ordinate, and the logarithm value of concentration of standard solution is abscissa, is understood after accompanying drawing 4 is changed, in 0.03 μ g/L~0.6
In μ g/L concentration ranges, logitW and aflatoxin B1Good linear relationship (see accompanying drawing 5) is presented in log concentration, must return
Equation Y=-2.852X-3.142, R2=0.980.
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=0.033, S=2.852, it is thus determined that test limit LOD is 0.038 μ g/L, quantitative limit
LOQ is 0.116 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 | 244.7 | Peach kernel | 329.4 | Spina date seed | 253.4 |
| The sterculia seed | 240.9 | Stiff silkworm | 270.2 | Centipede | 430.6 |
| Coix seed | 223.7 | Scorpio | 494.9 | Leech | 294.1 |
| Lotus seeds | 293.5 | The fruit of Rangoon creeper | 282.6 | Dried orange peel | 277.0 |
| Polygala | 451.1 | Cassia seed | 578.6 | Jujube | 392.5 |
| Betel nut | 272.5 | Earthworm | 341.4 | The seed of Oriental arborvitae | 590.6 |
(4) degree of accuracy (rate of recovery) and precision (repeatability)
Using the stiff silkworm without aflatoxin, lotus seeds, spina date seed, coix seed, peach kernel, leech as blank sample matrix, enter
The addition recovery test of three concentration levels of row, respectively with three batch kit measurements, calculate sample TIANZHU XINGNAO Capsul and batch in,
Interassay coefficient of variation, the results are shown in Table 7.
The sample veracity and precision result of table 7
As a result show, stiff silkworm, lotus seeds, spina date seed, coix seed, peach kernel, the TIANZHU XINGNAO Capsul of 6 kinds of matrix of leech 60%~
120%, crowd interior, the equal ＜ 15% of 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, AFs TIANZHU XINGNAO Capsuls are in normal range (NR).In view of having improper preservation during transport and use
Condition occurs, and kit is placed 8 days at 37 DEG C and carries out accelerated aging tests, as a result the indices of the kit are accorded with completely
Close and require；Situation is freezed in view of kit, kit is placed 8 days in -20 DEG C of refrigerators, as a result the kit is each
Item index is 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 anti-total aflatoxina monoclonal antibody, it is characterised in that：It is named as anti-total yellow
Aspertoxin monoclonal antibody hybridoma cell strain F-3-1, has been preserved in China Committee for Culture Collection of Microorganisms common
Microorganism center, deposit number is CGMCC No.13806, and preservation date is on 03 08th, 2017.
2. a kind of anti-total aflatoxina monoclonal antibody, it is characterised in that：It is to be as deposit number described in claim 1
What CGMCC No.13806 anti-total aflatoxina monoclonal antibody hybridoma cell strain F-3-1 secretions were produced.
3. the application of anti-total aflatoxina monoclonal antibody described in claim 2, it is characterised in that：For aspergillus flavus in Chinese medicine
Toxin B1、B2、G1、G2The analysis detection of total amount.
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