CN103743913B - Method for rapidly identifying host protein interacting with aflatoxin B1 - Google Patents
Method for rapidly identifying host protein interacting with aflatoxin B1 Download PDFInfo
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- CN103743913B CN103743913B CN201410031331.1A CN201410031331A CN103743913B CN 103743913 B CN103743913 B CN 103743913B CN 201410031331 A CN201410031331 A CN 201410031331A CN 103743913 B CN103743913 B CN 103743913B
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Abstract
The invention relates to a method for finding out a binding protein of aflatoxin B1 through solid phase chromatography. According to the method, the binding protein of the aflatoxin B1 can be rapidly and effectively found out, and research on toxication mechanisms of the aflatoxin B1 is promoted. More specifically, the method comprises the following steps: coupling the aflatoxin B1 to a carrier protein, fixing a coupling compound on a PVDF (Polyvinylidene Fluoride) film, incubating the coupling compound with total proteins, performing nonspecific washing and specific elution, and performing mass spectrum identification. According to in-vitro binding validation, the interacted protein, obtained in the method, of fungaltoxin is relatively high in accuracy.
Description
Technical field
The invention belongs to protein engineering field, be specifically related to one and screened and AFB by solid phase chromatography
1in conjunction with albumen method and to its whether with AFB
1there is the Validation in vitro analysis of associativity.
Background technology
Mycotoxin is that fungi grows produced metabolic product in food or feed, has high risks to humans and animals.Have over the years and in a large number pollute grain and crop about mycotoxin, cause the report of things poisoning, wherein common to mycotoxin have: aflatoxin, Citreoviridin, citrinin, T-2 toxin, the rare alcohol of deoxynivalenol bacterium etc.Wherein, aflatoxin (Aflatoxins, AFT) be primarily of aspergillus flavus (
aspergillus flavus) aspergillus parasiticus (
aspergillus parasiticus) secondary metabolite that produces, food mainly peanut, corn, paddy, wheat, the peanut wet goods oil and foodstuffs of pollution.It is 1 class carcinogenic substance that aflatoxin was delimited by the Agency for Research on Cancer of the World Health Organization (WHO) in 1993, and be the extremely strong extremely toxic substance of a kind of toxicity, its toxic action is mainly to the infringement of liver.With AFB in natural food
1(Aflatoxin B
1, AFB
1) the most common, harmfulness is also the strongest.Zoopery shows, AFB
1there is strong hepatotoxicity and carcinogenic effect.
Scientist is around AFB all the time
1product poison mechanism, toxin mechanism of toxication expand large quantity research, but due to this toxin be micromolecular feature, little by the study mechanism of animal Cell uptake for toxin.The present invention devises a kind of Rapid identification and AFB
1the method of the albumen of mutual work, the present invention finds out the associated proteins of aflatoxin by solid phase affinity chromatography, utilizes the method fast and effeciently to find out and AFB
1the associated proteins of mutual work, for promoting to lay the foundation to the research of the mechanism of toxication of this toxin.This method comprises, by AFB
1carry out coupling with carrier protein, the coupled complex of toxin and carrier protein is fixed on pvdf membrane, allow the total protein of coupled complex and host hatch, carry out non-specific washing and specificity wash-out and carry out Mass Spectrometric Identification.By the AFB that the method obtains
1interact protein accuracy high, can be laid the first stone by the research of animal Cell uptake mechanism for toxin.
Summary of the invention
The method of the host protein that the object of the present invention is to provide a kind of Rapid identification and aflatoxin B1 to do mutually, is screened and AFB by solid phase chromatography
1in conjunction with the method for albumen, and to itself and AFB
1whether there is the Validation in vitro analysis of associativity.Through screening and the checking of the method, have been found that at present 40S ribosomal protein SA and estradiol β dehydrogenasa 5 can and AFB
1in conjunction with, be AFB
1interact protein.
For achieving the above object, the present invention adopts following technical scheme:
A kind of Rapid identification and AFB
1the method of the host protein of mutual work is first by AFB
1coupling is carried out with bovine serum albumin(BSA), then by the method for solid phase chromatography, screening and AFB
1in conjunction with albumen, after mass spectral analyses, then by vitro binding assay checking albumen and AFB
1associativity.
The described method by solid phase chromatography is screened and AFB
1in conjunction with albumen, be by bovine serum albumin-AFB
1coupled complex is illustrated on the pvdf membrane after methyl alcohol activation, 3-5 DEG C of night incubation; Bovine serum albumin-AFB will have been hatched
1pvdf membrane massfraction be 2% bovine serum albumen solution carry out close after, film is placed in mouse liver total protein solution 3-5 DEG C of night incubation fully to combine, simultaneously with only showing that the pvdf membrane of bovine serum albumin molecule is placed in mouse liver total protein solution as negative control; Non-specific wash-out is carried out again with the phosphate buffer of 3-5 DEG C of precooling or phosphate Tween buffer, 3-5 DEG C of jolting 10min, wash four times, specificity wash-out is carried out with the NaCl of 2M, 3-5 DEG C of jolting 30min, carry out SDS-PAGE analysis after interact protein in eluent is concentrated, get differential band after silver dye and carry out mass spectral analyses.
Described verifies albumen and AFB by vitro binding assay
1associativity, to the AFB through Mass Spectrometric Identification
1after interact protein carries out gene clone, abduction delivering, purifying, by method validation corresponding protein and the AFB of ELISA
1associativity;
Described is verified by vitro binding assay, and concrete steps are: by bovine serum albumin-AFB
1coupled complex is coated in hole, spends the night at 3-5 DEG C, and the bovine serum albumen solution of 2wt.% after closed 2 h, adds the AFB of purifying at 37 DEG C
1interact protein 37 DEG C hatches 2 h, more in succession add primary antibodie and two resist, finally in succession add TMB colour developing and H
2sO
4stop buffer, arranges bag by the negative hole of bovine serum albumin in contrast simultaneously, verifies 40S ribosomal protein SA and estradiol β dehydrogenasa 5 and AFB through the method
1there is associativity.
Described primary antibodie is the His tag antibody of anti-albumen, hatches 1 h for 37 DEG C; Two goat anti-mouse IgG resisting the HRP for anti-His antibody to mark, hatch 1 h for 37 DEG C.
The invention has the advantages that: mycotoxin is that fungi grows produced metabolic product in food or feed, has high risks to humans and animals.Scientist is around mycotoxin all the time, particularly AFB
1mechanism of toxication expand large quantity research, but due to this toxin be micromolecular feature (being difficult to host protein that qualification is done mutually with it), for toxin by the mechanism of animal Cell uptake, and by little with the understanding of its interact protein in cell after Cell uptake.The invention provides a kind of truly feasible, and the method for fast and convenient screening and mycotoxin (for aflatoxin) interact protein, for the mechanism of toxication understanding mycotoxin further lays the foundation.
Accompanying drawing explanation
Fig. 1 AFB
1structural formula.
Fig. 2 AFB
1with BSA coupling route.
The UV scanning figure of Fig. 3 AFB1, BSA and coupled product three kinds of materials; Wherein 1 is bovine serum albumin; 2 is AFB
1; 3 is bovine serum albumin-AFB
1coupled complex.
The protein-bonded SDS-PAGE of Fig. 4 AFB1 analyzes; Wherein M is Marker; 1 is bovine serum albumin-AFB
1coupled complex; 2 is bovine serum albumin; 3 is bovine serum albumin-AFB
1coupled complex; 4 is bovine serum albumin; 1 and 2 swimming lanes are PBST washing groups, and 3 and 4 is PBS washing groups.
The AFB of Fig. 5 qualification
1protein-bonded vivoexpression and purifying.Wherein, A figure: the expression and purification of Rpsa albumen.M:Marker; 1:IPTG induces PET28a bacterium bacterium liquid; 2:IPTG induces PET28a-rpsa recombinant bacterium bacterium liquid; 3: the Rpsa albumen of purifying; B schemes: the expression and purification of Akr1c6 albumen.M:Marker; 1:IPTG induces PET28a bacterium bacterium liquid; 2:IPTG induces PET28a-akr1c6 recombinant bacterium bacterium liquid; 3: the Akr1c6 albumen of purifying; C schemes: the expression and purification of Cyb5a albumen.M:Marker; 1:IPTG induces PET28a bacterium bacterium liquid; 2:IPTG induces PET28a-cyb5a recombinant bacterium bacterium liquid; 3: the Cyb5a albumen of purifying.
The AFB of Fig. 6 qualification
1associated proteins and AFB
1external combination checking.Cross-linking products BSA-AFB1 is coated in enzyme mark hole, then adds albumen and primary antibodie (antibody of anti-albumen), two anti-, nitrite ion, the stop buffers of purifying, survey OD value, judged the associativity of aflatoxin B1 and albumen by OD value.
Embodiment
one, the preparation of polyacrylamide gel
(1) 2 mol/L Tris-HCl(pH8.8): take 24.2 g Tris base and add the dissolving of appropriate ultrapure water, with hydrochloric acid adjust pH to 8.8, add ultrapure water and be settled to 100 mL.
(2) 1 mol/L Tris-HCl(pH6.8): take 12.1 g Tris base and add the dissolving of appropriate ultrapure water, with hydrochloric acid adjust pH to 6.8, add ultrapure water and be settled to 100mL.
(3) 30 % acrylamide storage liquid: take 29.2 g acrylamides, 0.8 g N ', N '-Ya methylene diacrylamide, adds ultrapure water and is dissolved to 100 mL, and with Filter paper filtering after it dissolves completely, 4 DEG C keep in Dark Place.
(4) 10 % SDS: take 10 g SDS(electrophoresis levels), add ultrapure water and be dissolved to 100 mL, room temperature preservation.
(5) 10% ammonium persulfates: take 100 mg ammonium persulfates, add ultrapure water and are dissolved to 1 mL.
(6) 4 × separation gel damping fluids: measure 75 mL 2 mol/L Tris-HCl(pH8.8), 4 mL 10% SDS, add 21mL ddH2O, can preserve the several months at 4 DEG C.
(7) 4 × concentrated glue damping fluid: measure 50 mL 1 mol/L Tris-HCl(pH6.8), 4 mL 10% SDS, add 46 mL ddH2O, can preserve the several months at 4 DEG C.
(8) electrode buffer: take 3 g Tris base, 14.4 g glycocoll, 1 g SDS, adds appropriate ultrapure water and dissolves, with hydrochloric acid adjust pH to 8.3, add ultrapure water and be settled to 1000 mL.Also can be made into 10 × storage liquid, at room temperature can preserve for a long time.
(9) 5 × sample-loading buffers: take 10 mg bromophenol blues, add 0.6 mL 1 mol/L Tris-HCl(pH6.8), 2.5 mL glycerine, 2 mL 10 % SDS, 0.5 mL β-dredge base ethanol and 4.4 mL ddH2O, can preserve several weeks at 4 DEG C, or preserve the several months at-20 DEG C.
(10) Coomassie light blue dyeing liquor: take 1 g Coomassie light blue R-250,200 mL methyl alcohol, 50 mL glacial acetic acid, adding distil water is dissolved to 500 mL.
(11) Coomassie light blue destainer: measure 450 mL methyl alcohol, 100 mL glacial acetic acid, adding distil water to 1000
mL。
two, gene clone and protein purification reagent name and method
(1) 1.0% Ago-Gel: take 0.2 g agarose in 20 mL0.5 × TBE, heating for dissolving, be poured in glue plate, inserts comb,
(2) 5 × TBE: weigh 54.0 g Tris alkali, 27.5 g boric acid, add 800 mL, add the EDTA(pH8.0 of 20 mL 0.5 mol/L), after fully dissolving, use water constant volume to 1 L, room temperature storage.
(3) LB nutrient culture media: weigh 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, add 800 mL deionized waters, pH value is adjusted to 7.0, is settled to 1 L, after autoclave sterilization, 4 DEG C of preservations.
(4) ampicillin (Amp) (100 mg/mL): dissolve 1 g ampicillin sodium salt in enough water, be finally settled to 10 mL, filtration sterilization, be distributed into aliquot in-20 DEG C of storages.
(5) kanamycins (Kan) (50 mg/mL): dissolve 0.5 g kanamycins in enough water, be finally settled to 10 mL, filtration sterilization, is distributed into aliquot in-20 DEG C of storages.
(6) IPTG(isopropylthio-β-D-galactoside) (1 mol/L): dissolve the IPTG of 238 mg in 1 mL water, filtration sterilization, is stored in-20 DEG C.
(7) 0.1 mol/L CaCl2: take 54 g CaCl2-6H2O, solution is settled to 1 L by deionized water, degerming with 0.22 μm of frit, be distributed into 10 mL aliquots and be stored in-20 DEG C.
(8) Buffer B, C, D, E (L-1): take 13.8 g NaHPO4H2O, 1.2 g Tris Base, 480.5 g urea, after dissolving, regulate pH to 8.0 respectively, 6.3,5.9,4.5, use front adjust ph.
three, the title of each reagent and compound method in ELISA method
The carbonate bag of (1) 0.05 M is buffered liquid (pH 9.6): the sodium bicarbonate taking 1.59 g sodium carbonate and 2.93 g is respectively dissolved in the distilled water of 800 mL, regulate its pH value to 9.6 with the NaOH of 2 mol/L, be finally settled to 1000 mL.
(2) 1 × phosphate buffers (PBS): take NaCl 8.0 g, KCl 0.2 g, Na2HPO412H2O 3.58 g, KH2PO4 0.27 g, be dissolved in 1 L water, adjusts pH to 7.2 ~ 7.4,121 DEG C of autoclaving 20 min.
(3) 5%PBSM confining liquid: the skimmed milk power adding 4g in the 1 × PBS (pH 7.4) of 100mL.
(4) PBST eluent: namely containing Tween-20 concentration is the 1 × PBS of 0.05%.The Tween-20 of 0.5 mL is added in the 1 × PBS (pH 7.4) of 1000 mL.
(5) substrate colour developing A liquid: take 27.2 g sodium acetates and 3.2 g citric acids respectively in 1000 mL beakers, add the hydrogen peroxide of 30% of 0.6 mL, be finally settled to 1000 mL.
(6) substrate colour developing B liquid: take 0.4 g sodium ethylene diamine tetracetate, 1.9 g citric acids and 0.4 g tetramethyl benzidine (TMB) respectively in 1000 mL beakers, add 100 mL glycerine, be finally settled to 1000 mL.
(7) stop buffer (2 mol/L sulfuric acid): the concentrated sulphuric acid getting 111 mL instills slowly in the distilled water of 889 mL and mixes.
Below in conjunction with specific embodiment, the present invention is described in detail, and following examples are to further illustrate the present invention, but should not be considered as limiting the present invention.
Embodiment 1 Rapid identification and AFB
1host (small white mouse) albumen of mutual work
1) mycotoxin and carrier protein carry out coupling
AFB
1(Aflatoxin B
1be called for short AFB
1), be purchased from Simga company of the U.S..According to AFB
1design feature, adopt carbodlimide method carry out coupling.First carry out oximation reaction, get 2mg AFB
1be dissolved in 400 l pyridines with 4mg CMO, 25 DEG C of lucifuge jolting reaction 4h, namely obtain AFB by reaction product freeze drying
1oximated product; Get 0.2mg AFB
1oxime oximated product is dissolved in 100 μ L DMF-water (6:9 V/V), adds the mixing of 2mg EDC lucifuge, then adds 0.5% C-BSA solution, after 25 DEG C of lucifuges, 100r/min react 4h, then adds EDC 2mg continuation reaction 20h.Gained coupled product is loaded bag filter in 0.01mol/L PBS (pH7.4), put 4 DEG C of dialysis 3d, period changes dislysate.And to AFB
1, C-BSA and coupled product three kinds of materials carry out UV scanning, compare the change of coupled product absorption peak.
2) compound of coupling is fixed on pvdf membrane;
The PVDF membrane (pvdf membrane) of clip 10cm*1cm, after activating 5s, is washed by PBS solution, be placed in BSA-AFB in methanol solution
1in solution, shake slowly in 4 DEG C and spend the night, allow protein molecular and film fully be combined thus by BSA by AFB
1be illustrated in film surface.
3) coupled complex and total protein are hatched, coupled complex and total protein be combined with each other;
Extract Liver of Mice total protein by the epicyte protein of green skies company and suppressor proteins extraction agent box operation steps, survey protein concentration, and by PBS solution, extracted total protein is diluted to 2mg/ml.BSA-AFB will have been hatched
1pvdf membrane 2%BSA solution carry out closing after, get film and be placed in mouse liver total protein solution, shake overnight incubation slowly in 4 DEG C, allow protein molecular and AFB
1or BSA combines.Be placed in mouse liver total protein solution as negative control with only wrapping by the pvdf membrane of BSA molecule simultaneously.
4) carry out non-specific washing, specificity wash-out, obtain the albumen done mutually with mycotoxin.
PBS or the PBST damping fluid getting 4 DEG C of precoolings carries out non-specific washing, each 40mL, 4 DEG C of jolting 10min to the film after hatching, and washs four times.The PBS damping fluid containing 2M NaCl getting 4 DEG C of precoolings carries out specificity wash-out, 4 DEG C of jolting 30min to the film after washing, collects elute soln.Gained elute soln is loaded bag filter in 0.01mol/L PBS (pH7.4), put 4 DEG C of dialysis 2d, period changes dislysate, and concentrates the solution after dialysis with PEG20000.Then experimental group and control group are carried out further SDS-PAGE analysis, have 8 differential bands as seen after silver dye, tapped rubber and carry out mass spectral analyses.Concrete mass spectrophotometry condition is as follows:
LC condition: high performance liquid chromatograph: Thermo Scientific Accera System; Chromatographic column: BioBasic C18 Column (100 x 0.18 mm, particle size:5 um); Sample size: 10uL; Mobile phase: A: aqueous phase (0.1% formic acid); B: nitrile (0.1% formic acid); Gradient: 5% – 35%B in 20 minutes, 35% – 95%B in 2 minutes; Flow velocity: 2.5uL/min;
MS condition: mass spectrometer: LTQ-XL (Thermo Scientific); Spray voltage: 3.5 kV; Capillary temperature: 275 DEG C; Sheath gas velocity: 15arb; Precursor scans scope: 400-2000m/z; Isolation width:2 Da.
Second order ms condition: AGC Target 1e4,1 microscans; Collision energy: 35% CID.
Retrieval: the database that search uses is the Mus musculus.fasta protein pool downloaded from UNIPROT (http://www.uniprot.org/).The raw data Proteome Discoverer1.2 software that mass spectrophotometry obtains carries out relative quantitative assay.
5) mass spectral results analysis
Mass spectral results is analyzed, albumen that is inconsistent with corresponding stripe size and that mate peptide Duan Tai little is deleted, obtains 32 kinds of albumen; Again through document analysis, finally define three kinds of albumen of necessary checking further: 40S ribosomal protein SA(Rpsa) and estradiol β dehydrogenasa 5(Akr1c6), cytochrome b5 (Cyb5a).
Table 1 AFB
1the mass spectrophotometry of associated proteins band
6) AFB
1protein-bonded expression and purification
Extract mouse total serum IgE, after reverse transcription, obtain cDNA.The primer of design rpsa, akr1c6 and cyb5a gene, corresponding gene is cloned as template using the cDNA obtained, be building up on carrier PET28a, proceed to again in E.coli, add IPTG inducible protein to this bacterium after checking to express, then with nickel post, separation and purification is carried out to recombinant protein, obtain corresponding protein.
Table 1 primer sequence table
7) AFB
1associated proteins and AFB
1external combination checking
Enzyme-linked immunosorbent assay (ELISA) is adopted to analyze AFB
1the AFB obtained with qualification
1protein-bonded combination.Get cross-linking products BSA-AFB
1, being diluted to concentration with the carbonate buffer solution (pH 9.6) of 0.05M is 2 μ g/mL, and 100 μ L/ hole coated elisa plates, spend the night in 4 DEG C; PBS washes plate 3 times, and close with 2%BSA, 200 μ L/ holes, hatch 2 h in 37 DEG C; PBS washes plate 3 times, gets albumen Rpsa and Akr1c6 of purifying, and being diluted to concentration with 2%BSA is 10 μ g/mL, and 100 μ L/ holes add in enzyme mark hole; PBS washes plate 3 times, and press 1:4000 with 2%BSA and dilute HIS antibody, add ELISA Plate, 100 μ L/ holes, hatch 1 h in 37 DEG C; Respectively wash 3 times with PBST and PBS, press with 2%BSA the goat anti-mouse IgG that 1:6000 dilutes horseradish peroxidase mark, 100 μ L/ holes, hatch 1 h in 37 DEG C; Respectively wash 3 times with PBST and PBS, add TMB colour developing, 100 μ L/ holes, hatch 10 min in 37 DEG C; With 2 M H
2sO
4cessation reaction, 50 μ L/ holes; Survey 450nm light absorption value.BSA, trx and GFP albumen is set simultaneously as negative control, BSA-AFB
1cross-linking products, Rpsa albumen, Akr1c6 albumen, Cyb5a albumen are as positive control.Result display Rpsa albumen and Akr1c6 albumen and AFB
1there is associativity, but do not find Cyb5a albumen and AFB
1there is obvious adhesion.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University Of Agriculture and Forestry In Fujian
The method of the host protein that <120> Rapid identification and aflatoxin B1 are done mutually
<130> 6
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> artificial sequence
<400> 1
ataggatcca tgtccggagc cctt 24
<210> 2
<211> 25
<212> DNA
<213> artificial sequence
<400> 2
gccgaagctt tcaggaccac tcagt 25
<210> 3
<211> 29
<212> DNA
<213> artificial sequence
<400> 3
tacgaattca tggattctaa gcagcagac 29
<210> 4
<211> 26
<212> DNA
<213> artificial sequence
<400> 4
gccaagcttc cgttagtatt catccc 26
<210> 5
<211> 25
<212> DNA
<213> artificial sequence
<400> 5
taagaattca tggccgggca gtcag 25
<210> 6
<211> 28
<212> DNA
<213> artificial sequence
<400> 6
gccaagcttt caatcttctg ccatgtag 28
Claims (4)
1. a Rapid identification and AFB
1the method of the host protein of mutual work, is characterized in that: first by AFB
1coupling is carried out with bovine serum albumin(BSA), then by the method for solid phase chromatography, screening and AFB
1in conjunction with albumen, after mass spectral analyses, then by vitro binding assay checking albumen and AFB
1associativity;
The described method by solid phase chromatography is screened and AFB
1in conjunction with albumen, be by bovine serum albumin-AFB
1coupled complex is illustrated on the pvdf membrane after methyl alcohol activation, 3-5 DEG C of night incubation; Bovine serum albumin-AFB will have been hatched
1pvdf membrane massfraction be 2% bovine serum albumen solution carry out close after, film is placed in mouse liver total protein solution 3-5 DEG C of night incubation fully to combine, simultaneously with only showing that the pvdf membrane of bovine serum albumin molecule is placed in mouse liver total protein solution as negative control; Non-specific wash-out is carried out again with the phosphate buffer of 3-5 DEG C of precooling or phosphate Tween buffer, 3-5 DEG C of jolting 10-15min, wash three-four times, specificity wash-out is carried out with the NaCl of 2M, 3-5 DEG C of jolting 25-30min, carry out SDS-PAGE analysis after interact protein in eluent is concentrated, get differential band after silver dye and carry out mass spectral analyses.
2. a kind of Rapid identification according to claim 1 and AFB
1the method of the host protein of mutual work, is characterized in that: described verifies albumen and AFB by vitro binding assay
1associativity, to the AFB through Mass Spectrometric Identification
1after interact protein carries out gene clone, abduction delivering, purifying, by method validation corresponding protein and the AFB of ELISA
1associativity.
3. a kind of Rapid identification according to claim 2 and AFB
1the method of the host protein of mutual work, is characterized in that: described is verified by vitro binding assay, and concrete steps are: by bovine serum albumin-AFB
1coupled complex is coated in hole, spends the night at 3-5 DEG C, and the bovine serum albumen solution of 2wt.% after closed 2 h, adds the AFB of purifying at 37 DEG C
1interact protein 37 DEG C hatches 2 h, more in succession add primary antibodie and two resist, finally in succession add TMB colour developing and H
2sO
4stop buffer, arranges bag by the negative hole of bovine serum albumin in contrast simultaneously, verifies 40S ribosomal protein SA and estradiol β dehydrogenasa 5 and AFB through the method
1there is associativity.
4. a kind of Rapid identification according to claim 3 and AFB
1the method of the host protein of mutual work, is characterized in that: described primary antibodie is the His tag antibody of anti-albumen, hatches 1 h for 37 DEG C; Two goat anti-mouse IgG resisting the HRP for anti-His antibody to mark, hatch 1h for 37 DEG C.
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|---|---|---|---|---|
| CN87105153A (en) * | 1986-07-18 | 1988-02-03 | 宇部兴产株式会社 | The mensuration reagent and the assay method of mycotoxin |
| WO2003006994A1 (en) * | 2001-07-13 | 2003-01-23 | Diachemix Llc | Fluorescence polarization-based homogenous assay for aflatoxins |
| WO2006008143A2 (en) * | 2004-07-19 | 2006-01-26 | Grace Gmbh & Co. Kg | Apparatus components and methods of using apparatus components to detect the presence of an analyte |
| WO2007079893A1 (en) * | 2005-12-23 | 2007-07-19 | Bayer Technology Services Gmbh | Device and method for identifying mycotoxins |
-
2014
- 2014-01-23 CN CN201410031331.1A patent/CN103743913B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87105153A (en) * | 1986-07-18 | 1988-02-03 | 宇部兴产株式会社 | The mensuration reagent and the assay method of mycotoxin |
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