CN105457605A - Affinity adsorption material based on specific-binding aflatoxin nanobody - Google Patents

Affinity adsorption material based on specific-binding aflatoxin nanobody Download PDF

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CN105457605A
CN105457605A CN201510869030.0A CN201510869030A CN105457605A CN 105457605 A CN105457605 A CN 105457605A CN 201510869030 A CN201510869030 A CN 201510869030A CN 105457605 A CN105457605 A CN 105457605A
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aspergillus flavus
aflatoxin
chain antibody
sorbing material
heavy chain
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涂追
许杨
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Nanchang University
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Nanchang University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Abstract

The invention belongs to the field of biotechnology and relates to an immunoaffinity adsorption material based on a single-domain heavy-chain antibody, in particular to the immunoaffinity adsorption material based on aflatoxin. The immunoaffinity adsorption material comprises a vector and a ligand loaded on the vector, and is characterized in that the single-domain heavy-chain antibody specifically identifying the aflatoxin is taken as the ligand and has an amino acid sequence represented as SEQ ID NO: 1. The antibody has the characteristics of acid-base resistance, high temperature resistance, easiness in production and the like, and has important practical value for a low-cost re-usable immunological detection method of the aflatoxin.

Description

Based on the affine sorbing material of specific bond aflatoxin nano antibody
Technical field
The present invention relates to a kind of affine sorbing material of immunity based on single domain heavy chain antibody (also known as nano antibody technology), particularly for the affine sorbing material of immunity of aflatoxin.
Technical background
Aflatoxin is that a class is primarily of mycetogenetic hypertoxic secondary metabolites such as Aspergillus flavus, aspergillus parasiticus bacterium.Their chemical constitution is similar, is two furocoumarin derivatives, mainly comprises AFB 1(AFB 1), B 2(AFB 2), G 1(AFG 1), G 2(AFG 1) and M 1(AFM 1) etc.In the food and feed of natural contamination, AFB 1pollution the most common, its toxicity and carcinogenicity are also the strongest, are delimited as I class carcinogenic substance by the Agency for Research on Cancer of the World Health Organization.Countries in the world and area specify strict aflatoxin limit standard, such as, in European Union's infant food AFB 1allowance≤0.10 μ g/kg.
Existing Determination Methods of Aflatoxins mainly contains instrumental method, immunoassay and TLC.Wherein instrumental method has highly sensitive, accuracy and the advantage such as reproducible, but requires higher for analysis sample pre-treatments, needs to remove impurity in sample to reduce the interference to subsequent detection as far as possible.Traditional pre-treating method has liquid-phase extraction, SPE etc., and processing procedure is loaded down with trivial details and specificity is not high.Affinity chromatography technology, based on identification specific between aglucon and test substance, realizes the separation and purification to determinand in COMPLEX MIXED sample by single stepping.The aflatoxin affinity purification medium of current use generally adopts polyclonal antibody or monoclonal antibody as aglucon, there is not resistance to organic reagent, the active problem reduced rapidly.Therefore, the active novel aglucon that is high, good stability of exploitation is needed to replace conventional antibodies.Single domain heavy chain antibody is made up of the variable region of alpaca heavy chain antibody, is also called nano antibody, has acid and alkali-resistance, high temperature resistant and be easy to the characteristics such as production, has obvious advantage compared to conventional antibodies.
Summary of the invention
The object of this invention is to provide the affine sorbing material of a kind of immunity for aflatoxin and application thereof.
For achieving the above object, the present invention adopts following technical scheme:
Immunity for aflatoxin is affine, and sorbing material comprises carrier and aglucon, and described aglucon is single domain heavy chain antibody, has the amino acid sequence shown in SEQIDNO.:1.This aglucon can specific recognition aflatoxin.
Described aglucon can also on aforementioned single domain heavy chain antibody basis by random or site-directed mutagenesis technique carry out house of correction acquisition can with the antibody of aflatoxin specific binding.
Described carrier is magnetic bead, agarose gel microsphere, silica gel microball or porous material.
The preparation method of above-mentioned Aspergillus flavus toxin immuno is affine sorbing material, is characterized by:
When described carrier is magnetic bead, preparation method is: get 1mg carboxyl magnetic bead in centrifuge tube, adds 500 ~ 1000 μ l activation buffer (10mM, NaH 2pO 4, pH6.0), vortex mixed is even, and magnetic frame reclaims magnetic bead, then washs 2 times with activation buffer.Add 1 ~ 5mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS) respectively, after vortex mixed, leave standstill 30min.With coupling buffer (10mM, Na 2hPO 4, pH7.4) and wash magnetic bead 3 times, add aspergillus flavus resisting toxin single domain heavy chain antibody, room temperature reaction 2 ~ 6h, obtain the immunomagnetic beads of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody;
When described carrier is agarose gel microsphere, preparation method is: the dry glue 0.1MHCl activated by CNBr washs 10 ~ 15 times, balances 5 ~ 10min at every turn.With coupling buffer (10mM, Na 2hPO 4, pH7.4) and wash 5 ~ 15 times, add aspergillus flavus resisting toxin single domain heavy chain antibody, room temperature reaction 2 ~ 10h, obtain the affine sorbing material of immunity of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody;
When described carrier is silica gel microball, preparation method is: by silica gel microball pure water and phosphate buffer (PBS, 10mM, pH6.0) replace washing 5 ~ 10 times, with PBS buffer solution suspension silica gel microball, add aspergillus flavus resisting toxin single domain heavy chain antibody, mixing, adds the carbodiimide (EDC) of final concentration 1 ~ 10mg/ml, mixes rapidly, 4 DEG C of stirring reaction 12 ~ 24h, obtain the affine sorbing material of immunity of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody.
Affine for above-mentioned immunity sorbing material (carrier is agarose gel microsphere and silica gel microball) is filled to chromatographic column, namely Aspergillus flavus toxin immuno affinity column is obtained, method is: according to chromatographic column capacity, get the affine sorbing material of appropriate above-mentioned immunity in chromatographic column, add the PBS (10mM of 5 ~ 10 times of bed volumes, pH7.4), after washing, 4 DEG C, 20% ethanolic solution is stored in.
The immunomagnetic beads of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody is without the need to filling post, and directly after absorption, magnet is separated.
The invention still further relates to the affinity column being mounted with the affine sorbing material of described Aspergillus flavus toxin immuno.
The application of above-mentioned Aspergillus flavus toxin immuno is affine sorbing material, purifies sample extracting solution with the affine sorbing material of described Aspergillus flavus toxin immuno, enrichment aflatoxin (AFB 1, AFB 2, AFG 1or AFG 2).Aspergillus flavus toxin immuno is affine, and the application in aflatoxin, purification aflatoxin contamination material removed by sorbing material.This process is not for the purpose of medical diagnosis on disease treatment.When the carrier of immune absorption material be agarose gel microsphere and silica gel microball time, method is: first clean immune absorption material with pure water, add sample extracting solution, then pure water drip washing is used, use the aflatoxin of methanol-eluted fractions specific adsorption again, the eluent collected, is the sample extracting solution after purification and enrichment, can be used for subsequent analysis and detects.When carrier is magnetic bead, method is: added by immunomagnetic beads in pure water, and magnetic frame reclaims magnetic bead, repeated washing 3 ~ 5 times, then immunomagnetic beads is added in sample extracting solution, mixing, magnetic frame reclaims magnetic bead, after pure water cleans 1 ~ 3 time, the aflatoxin of methanol-eluted fractions specific adsorption, the eluent collected, is the sample extracting solution after purification and enrichment, can be used for subsequent analysis and detects.
The affine sorbing material aglucon of immunity that the present invention is directed to aflatoxin is single domain heavy chain antibody, and have the amino acid sequence shown in SEQIDNO.:1, this aglucon can specific recognition aflatoxin.This single domain heavy chain antibody easily obtains, and can produce aglucon by biological method mass propgation is single domain heavy chain antibody, avoids the loaded down with trivial details production methods such as artificial antibody, greatly reduces production cost.Production process, without the need to contacting aflatoxin, avoids producers' actual bodily harm.Have acid and alkali-resistance, high temperature resistant and be easy to the characteristics such as production, these characteristics have important practical value for the low cost of aflatoxin, reusable immunological detection method.
Detailed description of the invention
Below by the preparations and applicatio of the affine sorbing material of Aspergillus flavus toxin immuno, the present invention will be further described, and these specific embodiments should not be interpreted as limiting range of application of the present invention by any way.
Embodiment 1:
The structure of aspergillus flavus resisting toxin single domain heavy chain antibody (namely for the single domain heavy chain antibody of aflatoxin) non-immune libraries
By AFB 1with keyhole limpet hemocyanin (keyholelimpethemocyanin, KLH) covalent coupling, obtain aflatoxin artificial antigen AFB 1-KLH, gets 300 μ gAFB 1after-KLH and Freund's complete adjuvant emulsification, the immunity of subcutaneous multi-point injection is carried out to alpaca (Lamapacos).Booster immunization adopts 150 μ gAFB 1-KLH and incomplete Freund's adjuvant emulsification, interval is carried out for 2 weeks, and each immunity 7 days posterior veins get blood, adopts indirect elisa method to measure serum titer, selects the sample separation lymphocyte that serum titer is the highest, extract RNA.
The extraction of RNA is carried out with reference to TAKARA company RNAiso reagent description.Take RNA as template, oligodT is primer, with reference to TAKARA company reverse transcriptase description synthesis cDNA first chain.
Adopt PrimeSTAR high-fidelity DNA polymerase, obtain the variable region encoding gene (primer of employing is in table 1) of heavy chain antibody through nest-type PRC.First round PCR is respectively with primer AlpVh-LD and CH2-R amplification cDNA, and reaction condition is, 98 DEG C, 10s, 55 DEG C, 20s, 72 DEG C, 1min, 20 circulations, 98 DEG C, 10s, 68 DEG C, 1min, and 72 DEG C extend 10min.
By the first round PCR primer agarose gel electrophoresis of 1.2%, reclaim the DNA fragmentation of 600bp ~ 750bp, take turns the template of PCR as second, use primer AlpVh-SfiI and AlpVHHR1-NotI respectively, AlpVh-SfiI and AlpVHHR2-NotI, increases, and reaction condition is, 98 DEG C, 10s, 50 DEG C, 20s, 72 DEG C, 40s, 5 circulations, 98 DEG C, 10s, 68 DEG C, 40s, 30 circulations, 72 DEG C extend 10min.Reclaim kit through DNA fragmentation to reclaim, quantitatively, save backup in-20 DEG C.Phasmid pHEN1 and pcr amplification product are used SfiI, NotI double digestion respectively, reclaim through Ago-Gel, quantitatively after, with 1: 3 mol ratio, at 16 DEG C, connection of spending the night.
Table 1 library construction and identify primer used
Note: underscore represents restriction endonuclease recognition sequence
Connect product after alcohol settling, be dissolved in 10 μ L sterilized waters, divide and carry out Electroporation Transformation e. coli tg1 ten times.Get the bacterium liquid doubling dilution after 10 μ L electric shocks, cultivation, coating ampicillin 2 × YT culture plate, 37 DEG C, is inverted cultivation 12 ~ 16h, adopts primer M13-R and pHEN-R to carry out bacterium colony PCR, calculating storage capacity; Remainder all coats 24cm × 24cm ampicillin 2 × YT culture plate, 37 DEG C, is inverted cultivation 12 ~ 16h.With 10mL, after the lawn on culture plate scrapes by 2 × YT culture medium, add final concentration 15 ~ 30% glycerine, packing ,-80 DEG C save backup.
According to the storage capacity result calculated, the living cells of inoculation 10 times of storage capacities in the 2 × YT of 20mL (containing 2% glucose, 100 μ g/mL ampicillins), 30 DEG C, 220r/min is cultured to OD600 and reaches 0.5, adds helper phage by infection multiplicity 20: 1,37 DEG C, 220r/min, 60min.Culture is centrifugal, by 2 × YT (containing 100 μ g/mL ampicillins and 50 μ g/mL kanamycins) the resuspended precipitation of 50mL, 30 DEG C, after 220r/min incubated overnight, 3000g centrifuging and taking supernatant, add 5 × PEG/NaCl solution, place 1h or 4 DEG C on ice to spend the night, the centrifugal 30min of 12000rpm, resuspended phosphate buffer (PBS, the 0.01M be deposited in containing 10% glycerine, pH7.4), namely obtain aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries, get 10 μ L and measure titre, all the other are sub-packed in-80 DEG C and save backup.
Embodiment 2:
The elutriation of aspergillus flavus resisting toxin single domain heavy chain antibody and qualification
Adopt the method for solid phase affine elutriation from embodiment 1 gained aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries library elutriation for the single domain heavy chain antibody of aflatoxin.By AFB 1with oralbumin (albumin, OVA) covalent coupling, obtain artificial antigen AFB 1-OVA.Every hole adds the artificial antigen AFB that 100 μ L PBS dilute 1-OVA, 4 DEG C, wrap and spent the night, the bag of often taking turns elutriation is respectively 100 by concentration, 75,50 μ g/mL; Sucking-off coating buffer, PBS washes plate 3 times, and every hole adds 300 μ L3%BSA-PBS, 37 DEG C, closes 2h; PBS washes plate 6 times, adds 100 μ L phage antibody libraries (about containing 2 × 10 11cFU), 37 DEG C, 1.5h is hatched; The unconjugated bacteriophage of sucking-off, washes plate 5 times (by wheel increase by 5 times) with PBST (containing 0.5%Tween-20), then washes plate 10 times (washing plate number of times by wheel increase by 5 times) with PBS; With 100 μ L eluent (glycine-HCI, pH2.2) wash-out is adsorbed on the bacteriophage in enzyme mark hole, with in 50 μ LTris-HCl (1mol/L, pH8.0) and eluate, get 10 μ L for titer determination, for next round elutriation after the amplification of all the other eluates.Second takes turns and third round elutriation employing competitive elution, uses the AFB of 50 and 25ng/mL respectively 1solution, at 37 DEG C, hatches 1h.
After three-wheel elutriation, adopt the monoclonal of helper phage KM13 to random picking to rescue, obtain the phage particle showing antibody variable region respectively, binding activities and the specificity of phage particle is measured again with indirect phage-ELISA and indirect competition phage-ELISA, experiment setting negative control and ground control, concrete load procedure is in table 2.
Table 2 is phage-ELISA application of sample table indirectly
Send biotechnology service company to carry out sequencing ELISA positive colony, obtain the DNA sequence dna of Insert Fragment, it encodes for the single domain heavy chain antibody of aflatoxin, (SEQIDNO.:2) specific as follows:
CAGGTGCAGCTCGTGGAGTCGGGAGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGACTCTTTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACGACAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCACGTATTACTGTGCATTAGATAACCGCCGCAGTTATGTTGATTACCACTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
The amino acid sequence (SEQIDNO.:1) for the single domain heavy chain antibody of aflatoxin can be obtained according to DNA sequencing result and password sublist:
QVQLVESGGGLVQAGDSLRLSCAASGRTGTIYGMGWFREAPGKEREFVATLWWTVGAPYYADSVKGRFTISRDNDKNTVYLQMNSLKPEDTATYYCALDNRRSYVDYHSVSEYDYWGQGTQVTVSS
Indirect competition phage-ELISA method is adopted to measure, by AFB the cross reacting rate of positive colony from several different aflatoxin hypotype 1, AFB 2, AFG 1, AFG 2and AFM 1five kinds of standard items are diluted to 12 different working concentrations, carry out indirect competition phage-ELISA mensuration under identical condition, draw competitive ELISA curve respectively, standard concentration (IC when to calculate inhibiting rate be 50% 50), according to formula: cross reacting rate (%)=(AFB 1iC 50/ analog IC 50) × 100%, described analog is AFB 2, AFG 1, AFG 2or AFM 1, obtain positive colony of the present invention (the single domain heavy chain antibody for aflatoxin) for AFB 150% inhibition concentration.Result shows, positive colony of the present invention (the single domain heavy chain antibody for aflatoxin) is for AFB 1there is good specificity, to AFG 1and AFG 2also certain binding ability is had.
Embodiment 3:
The scale preparation of aspergillus flavus resisting toxin single domain heavy chain antibody
Encode anti-AFB 1the acquisition of the DNA fragmentation of single domain heavy chain antibody: 1. adopt restriction enzyme SfiI/NotI, the anti-AFB of double digestion phasmid pHEN- 1single domain heavy chain antibody genes, agarose gel electrophoresis reclaims anti-AFB 1single domain heavy chain antibody genes; 2. directly by anti-AFB 1single domain heavy chain antibody coded sequence send biotechnology service company to carry out chemical synthesis; 3. design Auele Specific Primer, increased from the cDNA storehouse that alpaca (Lamapacos) is originated by round pcr.
By the anti-AFB obtained 1single domain heavy chain antibody genes fragment is cloned into expression vector pET25, cuts qualification through PCR and enzyme, has built anti-AFB 1the colibacillus expression plasmid of single domain heavy chain antibody.
Expression plasmid is converted into e. coli bl21, and picking list bacterium colony carries out abduction delivering.By in single bacterium colony access 4mLLBA (Luria-Bertanibrothwith100 μ g/mLampicillin) fluid nutrient medium, 37 DEG C, 250r/min shaken cultivation 12h; Be transferred in 50mLLBA fluid nutrient medium with the inoculum concentration of 1% culture volume, 37 DEG C, 250r/min shaken cultivation is to OD 600reach 0.5 (about needing 2.5 ~ 3h), add the IPTG of final concentration 0.1mM, 30 DEG C, 200r/min Fiber differentiation.
Induced cultures 8000r/min is centrifugal, and in cell precipitation, add 20mL phosphate buffer (pH7.4) mixing, 8000r/min is centrifugal, removes supernatant, retains cell precipitation; 10mL same buffer is added in cell precipitation, mixing, ultrasonic cell-break process on ice, ultrasonication condition is 200W, broken 2s, interval 3s, totally 240 circulations, to the centrifugal 20min of clasmatosis thing 12000r/min at 4 DEG C, get supernatant and carry out affinitive layer purification and SDS-PAGE electrophoretic analysis, or in supernatant, adding the glycerine of final concentration 30%, mixing, is stored in-20 DEG C of refrigerator-freezers stand-by.
By optimizing abduction delivering condition (as Host Strains, expression vector, Fiber differentiation time, temperature and IPTG concentration etc.), destination protein (single domain heavy chain antibody) expression can be improved further, for preparing anti-AFB in a large number 1single domain heavy chain antibody provides approach.
The a small amount of preparation of embodiment 4 Aspergillus flavus toxin immuno is affine magnetic bead
Adopt nanometer magnetic bead as carrier, after coupling aspergillus flavus resisting single domain heavy chain antibody, obtain Aspergillus flavus toxin immuno magnetic bead, concrete preparation method is as follows:
The magnetic bead (transporting nanosecond science and technology Co., Ltd purchased from Wuxi hundred, carboxyl magnetic bead 300nm) getting 1mg carboxyl modified, in centrifuge tube, adds 500 μ l activation buffer (10mM, NaH 2pO 4, pH6.0), vortex mixed is even, and magnetic frame reclaims magnetic bead, then washs 2 times with activation buffer.Add 2mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS) respectively, after vortex mixed, leave standstill 30min.With coupling buffer (10mM, Na 2hPO 4pH7.4) magnetic bead is washed 3 times, add the aspergillus flavus resisting toxin single domain heavy chain antibody 1mg being dissolved in coupling buffer, room temperature reaction 3h, magnetic bead is washed 3 times with coupling buffer, add 500 μ l and close unreacted active group, room temperature reaction 30min containing the coupling buffer of 1% (w/v) bovine serum albumin(BSA) (BSA) or 1% (w/v) ovalbumin (OVA).Magnetic bead is washed 3 times, PBS solution (10mM, pH7.4,0.02%w/v, Na with coupling buffer 3n) 4 DEG C are stored in after resuspended.
The preparation of embodiment 5 Aspergillus flavus toxin immuno is affine sorbing material and affinity column
Adopt agarose microbeads as carrier, coupling aspergillus flavus resisting single domain heavy chain antibody, concrete preparation method is as follows:
The dry glue 0.1MHCl that CNBr activates is washed 10 times, balances 5min at every turn.With coupling buffer (10mM, Na 2hPO 4, pH7.4) and wash 10 times, add aspergillus flavus resisting toxin single domain heavy chain antibody (2mg/ every gram agarose microbeads), room temperature reaction 4h, make the agarose gel microsphere covalent coupling that aspergillus flavus resisting toxin single domain heavy chain antibody and CNBr activate.With coupling buffer (10mM, Na 2hPO 4, pH7.4) wash 2 times after, add confining liquid room temperature reaction 2h with close unreacted active group.The alternately washing 3 times of the phosphate buffer (10mM, pH7.4) amassed by colloid with 5 and acetate buffer solution (0.1M, pH4.0), obtains the affine sorbing material of immunity of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody.Getting the affine sorbing material of the above-mentioned immunity of 0.2ml is the chromatographic column of 1ml in capacity, after PBS (10mM, the pH7.4) washing of 5 ~ 10 times of bed volumes, adds 20% ethanolic solution, 4 DEG C of preservations.
The preparation of embodiment 6 Aspergillus flavus toxin immuno is affine sorbing material and affinity column
Adopt silica gel microball as carrier, coupling aspergillus flavus resisting single domain heavy chain antibody, concrete preparation method is as follows:
Get 2g silica gel microball pure water and phosphate buffer (PBS, 10mM, pH6.0) washing 5 ~ 10 times is replaced, with 10mlPBS buffer solution suspension silica gel microball, add 5mg aspergillus flavus resisting toxin single domain heavy chain antibody, mixing, add the carbodiimide (EDC) of final concentration 5mg/ml, rapid mixing, 4 DEG C of stirring reaction 12 ~ 24h, obtain the affine sorbing material of immunity of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody.Getting the affine sorbing material of the above-mentioned immunity of 0.2ml is the chromatographic column of 1ml in capacity, after PBS (10mM, the pH6) washing of 5 ~ 10 times of bed volumes, adds containing 0.02% (w/v) Na 3the PBS (10mM, pH6) of N, 4 DEG C of preservations.
Embodiment 7 Aspergillus flavus toxin immuno-affinity column column capacity, recovery of standard addition, reuse mensuration
Affinity column 5mlPBS (10mM, pH7.4) washing embodiment 5 or embodiment 6 prepared, adds the AFB that 5ml concentration is 100ng/ml 1standard solution, the drip washing of 10ml pure water, uses 1ml methanol-eluted fractions, collects eluent high performance liquid chromatography and detects AFB in eluent 1content, the capacity calculating affinity column is 650 ~ 800ng.
Get peanut, corn sample (not containing aflatoxin) that 5g pulverizes, add the AFB of 50ng, 100ng and 200ng respectively 1or AFG 1standard items, extract sample with 60% (v/v) methanol-water solution, vortex oscillation 15min, fast qualitative Filter paper filtering, gets 5ml filtrate, adds PBS (10mM, pH7.4) dilution.
Affinity column 5mlPBS (10mM, pH7.4) washing embodiment 5 or embodiment 6 prepared, add 10ml sample extraction dilution, the drip washing of 10ml pure water, uses 1ml methanol-eluted fractions, collects eluent high performance liquid chromatography and detects AFB 1or AFG 1content, calculate the rate of recovery.The affinity column of result display preparation is to AFB 1or AFG 1average recovery rate be respectively 85 ~ 101%, 80 ~ 97%.After reusing 10 times, affinity column is to AFB 1or AFG 1average recovery rate > 80%.

Claims (7)

1. the affine sorbing material of Aspergillus flavus toxin immuno, comprise carrier, be mounted in the aglucon on carrier, it is characterized in that this material is using the single domain heavy chain antibody of specific recognition aflatoxin as aglucon, the single domain heavy chain antibody of described specific recognition aflatoxin has the amino acid sequence shown in SEQIDNO.:1.
2. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, it is characterized in that, described aglucon on the amino acid sequence basis shown in SEQIDNO.:1 by random or site-directed mutagenesis technique carry out house of correction acquisition can with the antibody of aflatoxin specific binding.
3. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, is characterized in that, described carrier is magnetic bead.
4. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, is characterized in that, described carrier is Ago-Gel or silica gel microball or porous material.
5. be mounted with the affinity column of the affine sorbing material of Aspergillus flavus toxin immuno described in claim 1.
6. the application of the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1 in Aspergillus flavus toxin immuno detection, enrichment and purifying.
7. the application in aflatoxin, purification aflatoxin contamination material removed by the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1.
CN201510869030.0A 2015-12-02 2015-12-02 Affinity adsorption material based on specific-binding aflatoxin nanobody Pending CN105457605A (en)

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US6637438B1 (en) * 1997-04-21 2003-10-28 Kerry Scott Lane Method for assay and removal of harmful toxins during processing of tobacco products
CN103521181A (en) * 2013-10-10 2014-01-22 浙江丰虹新材料股份有限公司 Composite type mycotoxin adsorbent and preparation method thereof
CN103869065A (en) * 2014-03-28 2014-06-18 中国农业科学院油料作物研究所 Aflatoxin M1 nano antibody immunosorbent, immunoaffinity column, preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6637438B1 (en) * 1997-04-21 2003-10-28 Kerry Scott Lane Method for assay and removal of harmful toxins during processing of tobacco products
CN103521181A (en) * 2013-10-10 2014-01-22 浙江丰虹新材料股份有限公司 Composite type mycotoxin adsorbent and preparation method thereof
CN103869065A (en) * 2014-03-28 2014-06-18 中国农业科学院油料作物研究所 Aflatoxin M1 nano antibody immunosorbent, immunoaffinity column, preparation method and application thereof

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Application publication date: 20160406