CN102955031A - Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same - Google Patents
Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same Download PDFInfo
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Abstract
The invention provides an enzyme-linked immunosorbent assay kit for detecting an aflatoxin B1-containing medicine and an application for the same. The enzyme-linked immunosorbent assay (ELISA) kit comprises an ELISA plate coated with a coating antigen, an enzyme label, aflatoxin B1 specific antibody working solution (contained in the case that the coating antigen on the ELISA plate and the enzyme label are enzyme-labelled antibodies or enzyme-labelled antigens), aflatoxin B1 standard substance solution, substrate developing solution, stopping solution, concentrated washing solution and concentrated compound solution. The method for detecting aflatoxin B1 by virtue of the kit provided by the invention comprises the following steps of: performing sample pre-treatment at first, and then detecting by virtue of the kit, and finally analysing the detected result. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the residual amount of aflatoxin B1 in samples such as oil, peanuts and grains, as well as is simple and convenient to operate, low in expense, high in sensitivity, capable of being monitored in the field, and suitable for screening lots of samples.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of the aflatoxin B1 medicine and application thereof.
Background technology
Aflatoxin B1 (structural formula is seen Fig. 1) is the derivant of dihydrofuran coumarin, contains a bifuran and a coumarin, and the former is basic toxicity structure, and the latter is with carcinogenic relevant.Aflatoxin (B1, B2, G1, G2) is a group structural similarity, and by the deadly poisonous compound that aspergillus flavus, aspergillus parasiticus and A.nomius produce, aflatoxin can cause cancer, mainly is the pathology that causes liver, intestines, lung and chest.These fungis are born in food, feed and their raw material of Perenniporia martius, main cereal, rice, corn, beans, tree nut and the peanut etc. of polluting, cause serious threat for human health, the aflatoxin phenomenon that exceeds standard is all arranged in the import-export commodity in recent years, so aflatoxin has become the important object of entry and exit agricultural product security sanitary inspection.
European Union is given for animal feed or the human directly maximum residue limit(MRL) of the aflatoxin of consumable products is 2 μ g/L.For infant foods, infant's treating grain basis and infant nutrition food, maximum residue limit(MRL) is set as 0.1 μ g/L; China at the detectability of field of fodder at 4-40 μ g/L.
The detection method of aflatoxin B1, mainly contain thin-layer chromatography, high performance liquid chromatography, etc. multiple detection method.Thin-layered chromatography is to measure the classical way of aflatoxin, also is one of standard method of aflatoxin B1 in China's mensuration food and feeds, still, the thin-layered chromatography sample pre-treatments is loaded down with trivial details, and experimentation is complicated, and required time is long, poor accuracy is larger to experimenter's harm; The high performance liquid chromatography instrument and equipment is expensive, and technical merit is had relatively high expectations, and sample also needs to carry out purification process, is unfavorable for field screening, and the present invention has set up a kind of high sensitivity, and can the on-the-spot sampling rapid screening method of inspection.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of enzyme linked immunological kit that detects aflatoxin B1 is provided, enzyme linked immunological kit of the present invention is simple to operate, consuming time few, the fast detecting that can be used for aflatoxin B1 medicine in oil, peanut, the cereal sample, the screening that especially is fit to on-the-spot batch samples detects.
The present invention detects the enzyme linked immunological kit of aflatoxin B1, comprises coating antigen and enzyme labeling thing, and described coating antigen is selected from the conjugate of aflatoxin B1 haptens and carrier protein, aflatoxin b1 antibody and antiantibody; When described coating antigen was the conjugate of aflatoxin B1 haptens and carrier protein, described enzyme labeling thing was enzyme mark antiantibody; When described coating antigen was aflatoxin b1 antibody, described enzyme labeling thing was the conjugate of enzyme mark haptens and carrier protein; When described coating antigen was antiantibody, described enzyme labeling thing was the conjugate of enzyme mark aflatoxin B1 haptens and carrier protein; Described aflatoxin B1 haptens is with aflatoxin B1 and oxammonium hydrochloride condensation reaction, obtains by condensation reaction with succinic anhydride again.
Another object of the present invention provides the method for a kind of aflatoxin b1 antibody and this antibody of manufacture.Described aflatoxin b1 antibody has binding specificity to free or albumen etc. in conjunction with aflatoxin B1.Described aflatoxin b1 antibody can be monoclonal antibody or polyclonal antibody.
Aflatoxin B1 is small-molecule substance, only has immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response.In order to prepare specific antibody, must at first its structure of modification be obtained the aflatoxin B1 haptens, the haptenic synthetic method of aflatoxin B1 is to carry out structure of modification for the aflatoxin B1 molecule.The aflatoxin B1 haptens is with aflatoxin B1 and oxammonium hydrochloride condensation reaction, introduce a new hydroxyl, obtain having again the aflatoxin B1 haptens (Fig. 2) of carboxyl with the succinic anhydride reaction, this aflatoxin B1 haptens and macromolecular carrier albumen coupling obtain aflatoxin B1 comlete antigen (immunogene or coating antigen), and the antibody effect of its final preparation can embody in the aftermentioned invention.
Aforementioned bearer albumen can be ovalbumin, bovine serum albumin, mouse haemocyanin, thyroprotein, rabbit anteserum albumen, human albumin, hemocyanin or fibrinogen common carrier albumen.
The present invention adopts mixed anhydride method or carbodiimide method with aflatoxin B1 haptens and carrier protein couplet, has given prominence to the feature structure of aflatoxin B1, has increased the haptenic immunogenicity of aflatoxin B1.After measured among the present invention aflatoxin B1 haptens and bovine serum albumin(BSA) be 14～18: 1 in conjunction with mol ratio, be suitable for antibody preparation.
Described aflatoxin B1 specific antibody can be aflatoxin B1 monoclonal antibody or aflatoxin B1 polyclonal antibody; They all aforementioned aflatoxin B1 immunogen immune animal prepare.
Described aflatoxin B1 monoclonal antibody is preferably the aflatoxin B1 mouse monoclonal antibody.
Described aflatoxin B1 monoclonal antibody is preferably the monoclonal antibody of hybridoma cell strain D-2-2CGMCCNo.5130 secretion.
Described aflatoxin B1 monoclonal hybridoma strain D-2-2CGMCC No.5130 (Classification And Nomenclature: the strain of aflatoxin B1 monoclonal antibody hybridoma cell) be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 19th, 2011 and (be called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101).
Described aflatoxin B1 polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described aflatoxin B1 polyclonal antibody is preferably the aflatoxin B1 rabbit polyclonal antibody.
Starting material preparation process in the process of the present invention:
1. haptenic synthetic
Aflatoxin B1 haptens synthetic technology route such as Fig. 2.
2. the preparation of aflatoxin b1 antibody
(1) immunogene preparation
Adopt mixed anhydride method to carry out coupling aflatoxin B1 haptens and carrier protein and obtain immunogene.
(2) preparation of aflatoxin B1 mouse monoclonal antibody
A) animal immune program: adopt the Balb/c mouse as immune animal, take aflatoxin B1 haptens and carrier protein couplet thing as immunogene, after serum titer was greater than 1: 8000, the taking-up spleen carried out Fusion of Cells.
B) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and SP2/0 myeloma cell and merge, the screening cell line is until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
(3) preparation of aflatoxin B1 rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, as immunogene new zealand white rabbit is carried out immunity take aflatoxin B1 haptens and carrier protein couplet thing, repeatedly after the immunity, measure serum antibody titer.Wait tire higher after, put to death rabbit, gather serum, obtain polyclonal antibody.
3. the preparation of antiantibody
(1) the sheep anti mouse antiantibody be with sheep as immune animal, take mouse source antibody as immunogene anosis thalline sheep is carried out immunity, obtain the sheep anti mouse antiantibody;
(2) goat-anti rabbit antiantibody be with sheep as immune animal, take rabbit source antibody as immunogene anosis thalline sheep is carried out immunity, obtain goat-anti rabbit antiantibody.
4. Determination Methods of AFTB 1 provided by the present invention and detection kit thereof can be for detection of the aflatoxin B1 medicines in oil, peanut, the grain sample.
Described kit also comprises aflatoxin B1 standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.
Described aflatoxin B1 standard solution: 6 bottles, concentration is respectively 0,0.05,0.1,0.2,0.6 and 1.8 μ g/L.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; The sheep anti mouse antiantibody of enzyme labeling or goat-anti rabbit antiantibody adopt glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out coupling and obtain.
When marker enzyme is horseradish peroxidase, developer is comprised of nitrite ion A liquid and nitrite ion B liquid, nitrite ion A liquid is hydrogen peroxide or urea peroxide, and described nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme was alkaline phosphatase, nitrite ion was for being 1～2mol/L sodium hydroxide solution to nitro phosphate buffer, stop buffer.
Described concentrated cleaning solution is that the pH value is 7.2-7.5, contains 0.8-1.2% Tween-20,0.3-0.6 ‰ sodium azide, and the phosphate buffer of 0.1-0.2mol/L, described number percent are w/v.
Described concentrated redissolution liquid is pH7.5-7.8, contains the 2-4% casein, and the phosphate buffer of 0.1-0.2mol/L, described number percent are percent weight in volume.
Wherein ELISA Plate used coated damping fluid in preparation process is that the pH value is the carbonate buffer solution of 9.2-9.6,0.1-0.2mol/L, used confining liquid is for containing the 5-8% skimmed milk power, the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described number percent are w/v.
The preparation process of ELISA Plate is among the present invention: with coated damping fluid aflatoxin B1 coupled antigen, antibody or antiantibody are diluted to 0.15-0.25 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, coating buffer inclines, concentrated cleaning solution with dilution washs 2 times, each 30 seconds, get rid of liquid in the hole, behind thieving paper absorption residual moisture, in every hole, add 200 μ l confining liquids, 37 ℃ of incubation 2h, liquid in the hole of inclining, with the vacuum seal of aluminium film, 4 ℃ of dry places save backup after dry.
5. detection principle of the present invention is:
When pre-coated aflatoxin B1 coupled antigen on ELISA Plate, after adding sample solution or standard solution, add again aflatoxin B1 specific antibody solution, coated aflatoxin B1 coupled antigen competition aflatoxin B1 specific antibody on residual aflatoxin B1 medicine and the ELISA Plate in the sample, add the enzyme labeling antiantibody and carry out amplification, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, relatively can draw the residual quantity of aflatoxin B1 in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of aflatoxin B1 residual quantity in the judgement sample of the comparison of the aflatoxin B1 standard solution color of series concentration.
When pre-coated aflatoxin B1 specific antibody on ELISA Plate, after adding sample solution or standard solution, add again enzyme labeling aflatoxin B1 haptens solution, aflatoxin B1 medicine and enzyme-labelled antigen residual in the sample are competed the aflatoxin B1 specific antibody that is coated on the ELISA Plate, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, relatively can draw the residual content of aflatoxin B1 in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of aflatoxin B1 residual quantity in the judgement sample of the comparison of the aflatoxin B1 standard solution color of series concentration.
When pre-coated aflatoxin B1 coupled antigen on ELISA Plate, after adding sample solution or standard solution, add again enzyme labeling aflatoxin B1 specific antibody solution, coated aflatoxin B1 coupled antigen competition aflatoxin B1 specific antibody on residual aflatoxin B1 medicine and the ELISA Plate in the sample, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, relatively can draw the residual content of aflatoxin B1 in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of aflatoxin B1 residual quantity in the judgement sample of the comparison of the aflatoxin B1 standard solution color of series concentration.
When pre-coated antiantibody on ELISA Plate, after the adding aflatoxin b1 antibody is hatched, after adding sample solution or standard solution, add again enzyme labeling aflatoxin B1 coupled antigen solution, aflatoxin B1 medicine and enzyme labeling aflatoxin B1 coupled antigen residual in the sample are competed the aflatoxin B1 specific antibody, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, relatively can draw the residual content of aflatoxin B1 in the sample with typical curve.Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of aflatoxin B1 residual quantity in the judgement sample of the comparison of the aflatoxin B1 standard solution color of series concentration.
6. the present invention also provides a kind of method that detects the aflatoxin B1 medicine, and it comprises step:
(1) sample pre-treatments;
(2) detect with the aforementioned agents box;
(3) analyzing and testing result.
Determination mainly is for acquisition aflatoxin B1 solution from sample, thereby is used for follow-up detection.
When detecting with kit among the present invention: when coating antigen is the aflatoxin B1 coupled antigen, in the ELISA Plate micropore, add standard solution or sample solution, add ELIAS secondary antibody, add again antibody, after the washing, get rid of liquid in the hole behind the incubation, and absorb residual moisture with thieving paper, colour developing, termination are measured absorbance with microplate reader; When coating antigen is the aflatoxin B1 coupled antigen, add standard solution in the ELISA Plate micropore or sample solution adds enzymic-labelled antibody again, wash behind the incubation, get rid of liquid in the hole, and with thieving paper absorption residual moisture, colour developing, termination are measured absorbance with microplate reader; When coating antigen is the aflatoxin B1 specific antibody, add standard solution in the ELISA Plate micropore or sample solution adds enzyme labeling aflatoxin B1 haptens again, wash behind the incubation, get rid of liquid in the hole, and with thieving paper absorption residual moisture, colour developing, termination are measured absorbance with microplate reader; When coating antigen is antiantibody, in the ELISA Plate micropore, add aflatoxin b1 antibody, add enzyme mark aflatoxin B1 haptens after adding again standard solution or sample solution, wash behind the incubation, get rid of liquid in the hole, and with thieving paper absorption residual moisture, colour developing, termination are measured absorbance with microplate reader.
The Analysis of test results process is among the present invention: use the absorbance mean value (B) of standard solution of each concentration that obtains divided by the absorbance (B of first standard solution (0 standard)
0) multiply by again 100%, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Take the semilog value of the concentration (μ g/L) of aflatoxin B1 standard solution as X-axis, the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample then can be read from typical curve the residual quantity of aflatoxin B1 the sample.
The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.
Description of drawings
Fig. 1: aflatoxin B1 structural formula;
Fig. 2: aflatoxin B1 haptens synthetic technology route;
Fig. 3: kit typical curve.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
The preparation of embodiment 1 kit components
1. antigen is synthetic
A. haptenic synthetic
With aflatoxin B1 and oxammonium hydrochloride condensation reaction, introduce a new hydroxyl, obtain having again the haptens of carboxyl functional group with the succinic anhydride reaction.
Haptenic concrete steps: 156mg aflatoxin B1 and 105mg oxammonium hydrochloride were placed 5ml pyridine solution stirring at room 12-24 hour, revolve and boil off pyridine and unreacted oxammonium hydrochloride, obtain crude product, the crude product that obtains is added 5ml pyridine and 100mg succinic anhydride, and 60 ℃ were reacted 10-20 hour, revolved to steam pyridine, add 10ml water, ethyl acetate extraction, behind the evaporate to dryness in ethanol and water mixed system recrystallization, obtain the aflatoxin B1 haptens.
B. immunogene is synthetic
Aflatoxin B1 haptens and bovine serum albumin(BSA) are carried out coupling obtain immunogene.
Concrete behaviour is as follows:
Take by weighing 100mg BSA and be dissolved in 9.5ml PBS (0.01mol/L, the pH5.0) solution, take by weighing 50mg EDC and add, obtain I liquid; Take by weighing haptens 15mg and dissolve the rear I of adding liquid with 0.5mlDMF, stirring at room reaction 1h adds 50mg EDC again, and the dark place stirring reaction also spends the night; Change 3 times dislysate every day with 0.01mol/LPBS dialysis 3d.Packing saves backup in-20 ℃.
C. the preparation of coating antigen aflatoxin B1 coupled antigen
Aflatoxin B1 haptens and ovalbumin coupling are obtained coating antigen.
Concrete operations are as follows:
Get haptens 15mg and dissolve with 1.0mlDMF, get isobutyl chlorocarbonate 15 μ l and add, 10 ℃ of stirring reaction 30min can obtain reactant liquor A; Take by weighing OVA50mg, make it fully to be dissolved in the 6.0mL50mmol/L sodium carbonate liquor to get B liquid; Reactant liquor A dropwise slowly is added drop-wise among the reactant liquor B, 10 ℃ of reaction 4h, then 4 ℃ are spent the night; Change 3 times dislysate every day with 0.01mol/LPBS dialysis 3d.Packing saves backup in-20 ℃.
2. the preparation of monoclonal antibody
A. animal immune
Choosing healthy Balb/c mouse in 6-8 age in week, is that 100 μ g/ only carry out immunity according to immunizing dose.Get equivalent Freund's complete adjuvant (available from Sigma company, article No. F5881) during first immunisation and evenly mix with immunogene, during follow-up immunization and equivalent incomplete Freunds adjuvant (available from Sigma company, article No. F5506) mix.
B. Fusion of Cells and cloning
After the mice serum measurement result is higher, get its splenocyte, in 9: 1 ratio (quantity ratio) merge with SP2/0 myeloma cell, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
Obtain the monoclonal hybridoma strain D-2-2CGMCC No.5130 of aflatoxin B1 through screening.The antibody specificity of its secretion good (such as table 1), sensitivity can reach 0.05 μ g/L.
Antibody specific assay result falls in table 1D-2-2CGMCC No.5130 secretion Dan Ke
| Medicine name | Cross reacting rate (%) |
| |
100％ |
| AFB 2 | 193％ |
| Aflatoxin G 1 | 73％ |
| AFG 2 | 76％ |
C. cell cryopreservation and recovery
The monoclonal hybridoma strain of aflatoxin B1 is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. the production of monoclonal antibody and purifying
Get some of 6-8 Balb/c mouse in age in week, 0.5ml/ of Intraperitoneal injection sterilization paraffin oil, the monoclonal hybridoma strain 5 * 10 of 7 days pneumoretroperitoneums injection aflatoxin B1
7Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.
2. the preparation of polyclonal antibody
Adopt new zealand white rabbit as immune animal, take aflatoxin B1 haptens and bovine serum albumin(BSA) conjugate as immunogene, immunizing dose is 1.5mg/kg, Fu Shi with immunogene and equivalent when head exempts from helps (it is the same to originate) fully and is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape section is got the same dose immunogene and is added equivalent incomplete Freunds adjuvant (it is the same to originate) mixing and emulsifying in 3～4 weeks of interval, and booster immunization once, immunity is 5 times altogether, does not add for the last time adjuvant.Serum antibody titer is measured in last immunity afterwards blood sampling in 10 days, and the heart blood sampling obtains polyclonal antibody with sad-saturated ammonium sulfate method purifying.
3. the preparation process of sheep anti mouse antiantibody: as immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody; The preparation of goat-anti rabbit antiantibody: as immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take rabbit source antibody with sheep, obtain goat-anti rabbit antiantibody.
4. the preparation of ELISA Plate
With coated damping fluid aflatoxin B1 coupled antigen, antibody or antiantibody are diluted to 0.15-0.25 μ g/ml, every hole adds 100 μ l, and 37 ℃ of incubation 2h or 4 ℃ spend the night, coating buffer inclines, concentrated cleaning solution with dilution washs 2 times, each 30 seconds, gets rid of liquid in the hole, behind thieving paper absorption residual moisture, in every hole, add 200 μ l confining liquids, 37 ℃ of incubation 2h, liquid in the hole of inclining, with the vacuum seal of aluminium film, 4 ℃ of dry places save backup after dry.
Coating buffer is for the pH value is 9.6, the carbonate buffer solution of 0.1mol/L.
Confining liquid is for containing 8% skimmed milk power, and the phosphate buffer of pH value 7.4,0.2mol/L, described number percent are w/v.
5. the preparation of enzyme labeling sheep anti mouse antiantibody
Adopt the sodium periodate method after improveing to carry out coupling antiantibody and horseradish peroxidase (HR).The molar concentration rate of enzyme and antiantibody is 4: 1 in traditional sodium periodate method requirement reflection system; Because horseradish peroxidase produces many sites of being combined with antiantibody under the effect of strong oxidation, the horseradish peroxidase molecule of activation has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, make in the conjugate of preparation and be mixed with many condensates, in order to address this problem, we improve traditional method, that is:
1) saved amino closed process, because can produce self amino amino reality that connects seldom.
2) reduced horseradish peroxidase: the volumetric molar concentration ratio to 2 of antiantibody: 1, the method after the improvement
Easier than traditional method, the loss of the activity of enzyme is reduced.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of aflatoxin B1
Set up the enzyme linked immunological kit that detects aflatoxin B1, make it comprise following component:
(1) ELISA Plate of coated aflatoxin B1 coupled antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) aflatoxin B1 monoclonal antibody working fluid;
(4) the aflatoxin B1 standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.2 μ g/L, 0.6 μ g/L, 1.8 μ g/L;
(5) the substrate nitrite ion is comprised of A liquid and B liquid, and substrate nitrite ion A liquid is urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine;
(6) stop buffer is 2mol/L hydrochloric acid;
(7) concentrated cleaning solution is that the pH value is 7.4, contains the phosphate buffer of the 0.2mol/L of 1.0% Tween-20,0.4 ‰ sodium azide, and described number percent is w/v.
(8) the concentrated liquid that redissolves is pH7.6, contains 4% casein, and the phosphate buffer of 0.15mol/L, described number percent are w/v.
The detection of aflatoxin B1 in embodiment 3 actual samples
Sample pre-treatments
(1) oil (peanut oil, blending stock, corn oil, soya-bean oil equal samples)
Take by weighing 2.0 ± 0.05g edible oil sample to 50ml polystyrene centrifuge tube; Add 4ml methanol-water solution, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette the 2.0ml supernatant liquor to 50ml polystyrene centrifuge tube, add the 2.0ml deionized water, add again the 6ml methenyl choloride, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); The removal supernatant liquid is got 3ml lower floor organic phase to the clean glass test tube of 10ml, flows down in 50～60 ℃ of water-bath nitrogen to dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 30s dissolving dried residue, add 1ml and redissolve the liquid working fluid, with vortex instrument whirling motion 30s, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper strata normal hexane phase, take off layer water 50 μ l and be used for analyzing.
(2) peanut (living peanut, ripe peanut and salt peanut equal samples)
Take by weighing 2.0 ± 0.05g peanut sample to 50ml polystyrene centrifuge tube; Add the 3.0ml acetonitrile, add again the 3.0ml deionized water, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette the 3.0ml supernatant liquor to 50ml polystyrene centrifuge tube, add the 4.5ml methenyl choloride, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); The removal supernatant liquid is got 3ml lower floor organic phase to the clean glass test tube of 10ml, flows down in 50～60 ℃ of water-bath nitrogen to dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 30s dissolving dried residue, add 1ml and redissolve the liquid working fluid, with vortex instrument whirling motion 30s, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper strata normal hexane phase, take off layer water 50 μ l and be used for analyzing.
(3) cereal (analysis for soybean powder, rice meal, corn flour, flour equal samples)
Take by weighing 2.0 ± 0.05g cereal sample to 50ml polystyrene centrifuge tube; Add the 4.0ml acetonitrile, add again the 2.0ml deionized water, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette the 3.0ml supernatant liquor to 50ml polystyrene centrifuge tube, add the 6ml methenyl choloride, vibration 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); The removal supernatant liquid is got 4ml lower floor organic phase to the clean glass test tube of 10ml, flows down in 50～60 ℃ of water-bath nitrogen to dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 30s dissolving dried residue, add 1ml and redissolve the liquid working fluid, with vortex instrument whirling motion 30s, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper strata normal hexane phase, take off layer water 50 μ l and be used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with the aflatoxin B1 coupled antigen, add aflatoxin B1 standard solution or sample solution 50 μ l, add at random ELIAS secondary antibody 50 μ l, add again aflatoxin B1 monoclonal antibody working fluid 50 μ l, with cover plate mould shrouding, react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pour out liquid in the hole behind the 30s, repeat operation and wash altogether plate 5 times, remove residual moisture with thieving paper at last, every hole adds substrate nitrite ion A liquid urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine (TMB), the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds 2mol/L stop buffer hydrochloric acid 50 μ l, the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. Analysis of test results
With the absorbance mean value (B) of the standard solution of each concentration that the obtains absorbance (B divided by first standard solution (0 standard)
0) multiply by again 100%, obtain the percentage absorbance.Take the semilog value of aflatoxin B1 standard items concentration (μ g/L) as X-axis, the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, then can read from typical curve the residual quantity of aflatoxin B1.
The mensuration of embodiment 4 kit quality
1 standard items precision test:
Respectively extract a collection of ELISA Plate out respectively from the ELISA Plate of three different time period preparations, every batch is extracted 10 kits, and every plate is extracted 20 micropores out, measures the absorbance of 0.2 μ g/L standard solution, calculates the coefficient of variation.
The repeatable test of table 2 standard (CV%)
Can draw by above-mentioned test findings, every batch of each 10 standard items coefficient of variation of kit meet precision and are less than or equal to 25% regulation between 2.4%-8.1%.
The test of 2 sample preci-sion and accuracies
A) sample precision test:
Aflatoxin B1 with 0.2 μ g/L concentration is added mensuration to oil, peanut, cereal, gets respectively the kit of three different batches, and each concentration repeats 5 times and measures, and calculates respectively the coefficient of variation, the results are shown in Table 3.
The test of this repeatability of table 3 oil sample
The repeatable test of table 4 peanut sample
The repeatable test of table 5 cereal sample
The result shows the variation lines number average of oil, peanut, cereal sample between 7.3%-10.8%, has met that " " Ministry of Agriculture's file " farming doctor sends out [2005] No. 17 annex 2 kits and puts on record with reference to the 4th precision standard in the judgment criteria.
B) sample accuracy test
The aflatoxin B1 standard solution of getting two concentration is respectively 0.2 μ g/kg, 0.4 μ g/kg, respectively sample is added recovery test, each concentration do 4 parallel, accuracy in computation.
The accuracy of table 6 kit
The result shows that oil, peanut, cereal sample add the recovery between 76.9%-87.9%.Having met " Ministry of Agriculture's file " farming doctor [2005] No. 17 annex 2 kits puts on record with reference to standard of accruacy in the judgment criteria.
3 kits are preserved experiment
The kit preservation condition is 2～8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, aflatoxin B1 were added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use procedure, and kit was placed 12 days under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that this kit indices meets the requirements fully.Consider that the freezing situation of kit occurs, kit was put into-20 ℃ of refrigerator freezings 5 days, measurement result shows that also the kit indices is fully normal.Can draw kit from above result can preserve more than 12 months at 2～8 ℃.
Claims (9)
1. an enzyme linked immunological kit that detects aflatoxin B1 comprises coating antigen and enzyme labeling thing, and described coating antigen is selected from conjugate, aflatoxin b1 antibody and the antiantibody of aflatoxin B1 haptens and carrier protein; When described coating antigen was the conjugate of AFB 1 haptens and carrier protein, described enzyme labeling thing was enzyme mark antiantibody; When described coating antigen was aflatoxin b1 antibody, described enzyme labeling thing was the conjugate of enzyme mark haptens and carrier protein; When described coating antigen was antiantibody, described enzyme labeling thing was the conjugate of enzyme mark aflatoxin B1 haptens and carrier protein; Described AFB 1 haptens is with aflatoxin B1 and oxammonium hydrochloride condensation reaction, obtains by condensation reaction with succinic anhydride again.
2. enzyme linked immunological kit according to claim 1 is characterized in that described aflatoxin b1 antibody is the aflatoxin B1 monoclonal antibody, is that the conjugate with aflatoxin B1 haptens and carrier protein prepares as immunogene; Described carrier protein is ovalbumin, bovine serum albumin, mouse haemocyanin, thyroprotein, rabbit anteserum albumen, human albumin, hemocyanin or fibrinogen.
3. enzyme linked immunological kit according to claim 1 and 2 is characterized in that described aflatoxin B1 monoclonal antibody is produced by hybridoma cell strain D-2-2CGMCC No.5130 secretion.
4. enzyme linked immunological kit according to claim 1 and 2, the marker enzyme that it is characterized in that described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase.
5. enzyme linked immunological kit according to claim 1 and 2 is characterized in that described antiantibody is the sheep anti mouse antiantibody.
6. enzyme linked immunological kit according to claim 1 and 2 is characterized in that described kit also comprises aflatoxin B1 standard solution, nitrite ion, concentrated cleaning solution, concentrated liquid, the stop buffer of redissolving; Described concentrated cleaning solution is that the pH value is 7.2-7.5, contains 0.8-1.2% Tween-20,0.3-0.6 ‰ sodium azide, the phosphate buffer of 0.1-0.2mol/L; Described concentrated redissolution liquid is pH7.5-7.8, contains the 2-4% casein, the phosphate buffer of 0.1-0.2mol/L; Described substrate nitrite ion is comprised of nitrite ion A liquid and nitrite ion B liquid, and nitrite ion A liquid is hydrogen peroxide or urea peroxide, and nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine; Described stop buffer is 1～2mol/L sulfuric acid or hydrochloric acid solution; Described coated damping fluid is that the pH value is the carbonate buffer solution of 9.2-9.6,0.1-0.2mol/L; Described confining liquid is for containing the 5-8% skimmed milk power, and the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described number percent are w/v.
7. enzyme linked immunological kit according to claim 4, it is characterized in that when marker enzyme is horseradish peroxidase, described developer is comprised of nitrite ion A liquid and nitrite ion B liquid, nitrite ion A liquid is hydrogen peroxide or urea peroxide, nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1～2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was the nitro phosphate buffer, and stop buffer is 1～2mol/L sodium hydroxide solution.
8. a method that detects aflatoxin B1 may further comprise the steps;
(1) sample pre-treatments;
(2) detect with each described enzyme linked immunological kit of claim 1-7;
(3) analyzing and testing result.
9. preserving number is the aflatoxin B1 monoclonal antibody hybridoma cell strain D-2-2 of CGMCC No.5130.
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