CN102955031B - 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 enzyme linked immunosorbent detection technology, being specifically related to a kind of enzyme linked immunological kit for detecting aflatoxin B1 medicine and application thereof.
Background technology
Aflatoxin B1 (structural formula is shown in Fig. 1) is the derivant of dihydrofuran coumarin, and containing a bifuran and a coumarin, the former is basic toxin moiety, and the latter is relevant with carcinogenic.Aflatoxin (B1, B2, G1, G2) is that a group structure is similar, and the deadly poisonous compound produced by aspergillus flavus, aspergillus parasiticus and A.nomius, aflatoxin can cause cancer, mainly causes the pathology of liver, intestines, lung and chest.These fungis are born in the food of Perenniporia martius, feed and their raw material, main pollution cereal, rice, corn, beans, tree nut and peanut etc., serious threat is caused to the health of the mankind, in recent years all have aflatoxin to exceed standard phenomenon in import-export commodity, therefore aflatoxin has become the important object of entry and exit agricultural product security sanitary inspection.
The maximum residue limit(MRL) that European Union is given for the aflatoxin of animal feed or the direct consumable products of the mankind is 2 μ g/L.For infant foods, infant's treating grain basis and infant nutrition foods, 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 the classical way measuring aflatoxin, and Ye Shi China measures one of standard method of aflatoxin B1 in food and feeds, but, 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; High performance liquid chromatography instrument and equipment is expensive, and technical merit requires higher, and sample also needs to carry out purification process, is unfavorable for field screening, and the present invention establishes 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 detecting aflatoxin B1 is provided, enzyme linked immunological kit of the present invention is simple to operate, consuming time few, can be used for the quick detection of aflatoxin B1 medicine in oil, peanut, cereal sample, be especially applicable to the selective mechanisms of on-the-spot batch samples.
The present invention detects the enzyme linked immunological kit of aflatoxin B1, comprises coating antigen and enzyme marker, 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 is the conjugate of aflatoxin B1 haptens and carrier protein, described enzyme marker is enzyme mark antiantibody; When described coating antigen is aflatoxin b1 antibody, described enzyme marker is the conjugate of enzyme mark haptens and carrier protein; When described coating antigen is antiantibody, described enzyme marker is the conjugate of enzyme mark aflatoxin B1 haptens and carrier protein; Described aflatoxin B1 haptens is by aflatoxin B1 and oxammonium hydrochloride condensation reaction, then obtained by condensation reaction with succinic anhydride.
Another object of the present invention is to provide a kind of method 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, first its structure of modification must be obtained aflatoxin B1 haptens, the haptenic synthetic method of aflatoxin B1 carries out structure of modification for aflatoxin B1 molecule.Aflatoxin B1 haptens is with aflatoxin B1 and oxammonium hydrochloride condensation reaction, introduce a new hydroxyl, the aflatoxin B1 haptens (Fig. 2) with carboxyl is obtained by reacting again with succinic anhydride, this aflatoxin B1 haptens and macromolecular carrier albumen coupling obtain aflatoxin B1 comlete antigen (immunogene or coating antigen), and its antibody effects finally prepared can embody in aftermentioned invention.
Aforementioned bearer albumen can be ovalbumin, bovine serum albumin, mouse haemocyanin, thyroprotein, rabbit serum proteins, human albumin, hemocyanin or fibrinogen common carrier albumen.
The present invention adopts mixed anhydride method or carbodiimide method by aflatoxin B1 haptens and carrier protein couplet, highlights the feature structure of aflatoxin B1, adds the haptenic immunogenicity of aflatoxin B1.After measured in 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; Their all aforementioned aflatoxin B1 immunogen immune animals prepare.
Described aflatoxin B1 monoclonal antibody is preferably aflatoxin B1 mouse monoclonal antibody.
Described aflatoxin B1 monoclonal antibody is preferably the monoclonal antibody that hybridoma cell strain D-2-2CGMCCNo.5130 secretes.
Described aflatoxin B1 monoclonal hybridoma strain D-2-2CGMCC No.5130 (Classification And Nomenclature: aflatoxin B1 monoclonal antibody hybridoma cell strain) has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 08 19th, 2011, 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 aflatoxin B1 rabbit polyclonal antibody.
Starting material preparation process in process of the present invention:
1. haptenic synthesis
Aflatoxin B1 hapten synthesis technology path is 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 Balb/c mouse as immune animal, with aflatoxin B1 haptens with carrier protein couplet thing for immunogene, after serum titer is greater than 1: 8000, taking-up spleen carries out Fusion of Cells.
B) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and SP2/0 myeloma cell fusion, screening cell line, 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, for immunogene, immunity is carried out to new zealand white rabbit with aflatoxin B1 haptens and carrier protein couplet thing, repeatedly after immunity, measure serum antibody titer.Wait tire higher after, put to death rabbit, gather serum, obtain polyclonal antibody.
3. the preparation of antiantibody
(1) sheep anti mouse antiantibody is using sheep as immune animal, carries out immunity, obtain sheep anti mouse antiantibody with mouse source antibody for immunogene to anosis thalline sheep;
(2) goat-anti rabbit antiantibody is using sheep as immune animal, carries out immunity with rabbit source antibody for immunogene to anosis thalline sheep, obtains goat-anti rabbit antiantibody.
4. Determination Methods of AFTB 1 provided by the present invention and detection kit thereof may be used for detecting the aflatoxin B1 medicine in oil, peanut, 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 marker 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 Over-voltage protection that marker enzyme and antiantibody are carried out coupling and obtain.
When marker enzyme is horseradish peroxidase, developer is made up 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 is alkaline phosphatase, nitrite ion is for being 1 ~ 2mol/L sodium hydroxide solution to nitro phosphate buffer, stop buffer.
Described concentrated cleaning solution is pH value is 7.2-7.5, containing 0.8-1.2% Tween-20,0.3-0.6 ‰ sodium azide, the phosphate buffer of 0.1-0.2mol/L, described number percent is w/v.
Described concentrated redissolution liquid is pH7.5-7.8, and containing 2-4% casein, the phosphate buffer of 0.1-0.2mol/L, described number percent is percent weight in volume.
The carbonate buffer solution of the bag that wherein ELISA Plate is used in preparation process is buffered liquid to be pH value be 9.2-9.6,0.1-0.2mol/L, confining liquid used is for containing 5-8% skimmed milk power, the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described number percent is w/v.
In the present invention, the preparation process of ELISA Plate is: be buffered liquid with bag and aflatoxin B1 coupled antigen, antibody or antiantibody are diluted to 0.15-0.25 μ g/ml, every hole adds 100 μ l, 37 DEG C of incubation 2h or 4 DEG C spend the night, incline coating buffer, 2 times are washed with the concentrated cleaning solution of dilution, each 30 seconds, get rid of liquid in hole, after absorbing residual moisture with thieving paper, 200 μ l confining liquids are added, 37 DEG C of incubation 2h, liquid in hole of inclining in every hole, with the vacuum seal of aluminium film after dry, 4 DEG C of dry places save backup.
5. Cleaning Principle of the present invention is:
When in ELISA Plate during pre-coated aflatoxin B1 coupled antigen, after adding sample solution or standard solution, add aflatoxin B1 specific antibody solution again, aflatoxin B1 coupled antigen aflatoxin B1 medicine residual in sample and ELISA Plate being wrapped quilt competes aflatoxin B1 specific antibody, add 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, compares the residual quantity that can draw aflatoxin B1 in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, can the concentration range of aflatoxin B1 residual quantity in judgement sample roughly with comparing of the aflatoxin B1 standard solution color of series concentration.
When in ELISA Plate during pre-coated aflatoxin B1 specific antibody, after adding sample solution or standard solution, add enzyme labeling aflatoxin B1 haptens solution again, aflatoxin B1 medicine residual in sample and enzyme-labelled antigen compete the aflatoxin B1 specific antibody be coated in ELISA Plate, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, compares the residual content that can draw aflatoxin B1 in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, can the concentration range of aflatoxin B1 residual quantity in judgement sample roughly with comparing of the aflatoxin B1 standard solution color of series concentration.
When in ELISA Plate during pre-coated aflatoxin B1 coupled antigen, after adding sample solution or standard solution, add enzyme labeling aflatoxin B1 specific antibody solution again, aflatoxin B1 coupled antigen aflatoxin B1 medicine residual in sample and ELISA Plate being wrapped quilt competes aflatoxin B1 specific antibody, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, compares the residual content that can draw aflatoxin B1 in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, can the concentration range of aflatoxin B1 residual quantity in judgement sample roughly with comparing of the aflatoxin B1 standard solution color of series concentration.
When in ELISA Plate during pre-coated antiantibody, add after aflatoxin b1 antibody hatches, after adding sample solution or standard solution, add enzyme labeling aflatoxin B1 coupled antigen solution again, aflatoxin B1 medicine residual in sample and enzyme labeling aflatoxin B1 coupled antigen compete aflatoxin B1 specific antibody, develop the color with nitrite ion, the content of sample light absorption value and aflatoxin B1 medicine is negative correlation, compares the residual content that can draw aflatoxin B1 in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, can the concentration range of aflatoxin B1 residual quantity in judgement sample roughly with comparing of the aflatoxin B1 standard solution color of series concentration.
6. present invention also offers a kind of method detecting aflatoxin B1 medicine, it comprises step:
(1) sample pre-treatments;
(2) detect with aforementioned agents box;
(3) testing result is analyzed.
The pre-treatment of sample mainly in order to obtain aflatoxin B1 solution from sample, thus for follow-up detection.
When detecting with kit in the present invention: when coating antigen is aflatoxin B1 coupled antigen, standard solution or sample solution is added in ELISA Plate micropore, add ELIAS secondary antibody, add antibody again, after incubation after washing, get rid of liquid in hole, and absorb residual moisture with thieving paper, colour developing, termination, measure absorbance by microplate reader; When coating antigen is aflatoxin B1 coupled antigen, in ELISA Plate micropore, add standard solution or sample solution adds enzymic-labelled antibody again, wash after incubation, get rid of liquid in hole, and absorb residual moisture with thieving paper, colour developing, termination, measure absorbance by microplate reader; When coating antigen is aflatoxin B1 specific antibody, standard solution is added or sample solution adds enzyme labeling aflatoxin B1 haptens again in ELISA Plate micropore, wash after incubation, get rid of liquid in hole, and absorb residual moisture with thieving paper, colour developing, termination, measure absorbance by microplate reader; When coating antigen is antiantibody, aflatoxin b1 antibody is added in ELISA Plate micropore, enzyme mark aflatoxin B1 haptens is added after adding standard solution or sample solution again, wash after incubation, get rid of liquid in hole, and absorb residual moisture with thieving paper, colour developing, termination, measure absorbance by microplate reader.
In the present invention Analysis of test results process for: with the absorbance values (B) of the standard solution of each concentration the obtained absorbance (B divided by first standard solution (0 standard)
0) be multiplied by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) × 100%
With the semilog value of the concentration of aflatoxin B1 standard solution (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of each sample corresponding then can read the residual quantity of aflatoxin B1 in sample from typical curve.
In the present invention, the analysis of testing result also can adopt regression equation method, calculates sample solution concentration.
Accompanying drawing explanation
Fig. 1: aflatoxin B1 structural formula;
Fig. 2: aflatoxin B1 hapten synthesis technology path;
Fig. 3: kit standard curve.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
The preparation of embodiment 1 reagent constituents
1. the synthesis of antigen
A. haptenic synthesis
By aflatoxin B1 and oxammonium hydrochloride condensation reaction, introduce a new hydroxyl, then be obtained by reacting the haptens with carboxyl functional group with succinic anhydride.
Haptenic concrete steps: 156mg aflatoxin B1 and 105mg oxammonium hydrochloride are placed in 5ml pyridine solution stirring at room temperature 12-24 hour, revolve and boil off pyridine and unreacted oxammonium hydrochloride, obtain crude product, the crude product obtained is added 5ml pyridine and 100mg succinic anhydride, 60 DEG C of reaction 10-20 hour, revolve and steam pyridine, add 10ml water, extraction into ethyl acetate, after evaporate to dryness in ethanol and water mixed system recrystallization, obtain aflatoxin B1 haptens.
B. immunogene synthesis
Aflatoxin B1 haptens and bovine serum albumin(BSA) are carried out coupling and obtains immunogene.
Concrete behaviour is as follows:
Take 100mg BSA to be dissolved in 9.5ml PBS (0.01mol/L, pH5.0) solution, take 50mg EDC and add, obtain I liquid; Take after haptens 15mg 0.5mlDMF dissolves and add I liquid, stirring at room temperature reaction 1h, then add 50mg EDC, dark place stirring reaction also spends the night; 3 dislysates are changed every day with 0.01mol/LPBS dialysis 3d.Packing, saves backup in-20 DEG C.
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 1.0mlDMF to dissolve, get isobutyl chlorocarbonate 15 μ l and add, 10 DEG C of stirring reaction 30min, can obtain reactant liquor A; Take OVA50mg, to make it fully to be dissolved in 6.0mL50mmol/L sodium carbonate liquor to obtain B liquid; Dropwise slowly be added drop-wise in reactant liquor B by reactant liquor A, 10 DEG C of reaction 4h, then 4 DEG C are spent the night; 3 dislysates are changed every day with 0.01mol/LPBS dialysis 3d.Packing, saves backup in-20 DEG C.
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, article No. F5881) and immunogene Homogeneous phase mixing during first immunisation, during follow-up immunization and equivalent incomplete Freund's adjuvant (available from Sigma, article No. F5506) mix.
B. Fusion of Cells and cloning
After mice serum measurement result is higher, get its splenocyte, in 9: 1 ratios (number ratio) and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
The monoclonal hybridoma strain D-2-2CGMCC No.5130 of aflatoxin B1 is obtained through screening.The antibody specificity good (as table 1) of its secretion, sensitivity can reach 0.05 μ g/L.
Table 1D-2-2CGMCC No.5130 secretes Dan Ke and falls antibody specific assay result
| Medicine name | Cross reacting rate (%) |
| Aflatoxin B1 | 100％ |
| AFB 2 | 193％ |
| Aflatoxin G 1 | 73％ |
| AFG 2 | 76％ |
C. cell cryopreservation and recovery
The monoclonal hybridoma strain cryopreserving liquid of aflatoxin B1 is made 1 × 10
9the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
D. the production of monoclonal antibody and purifying
Get 6-8 Balb/c mouse in age in week some, only, within 7 days, pneumoretroperitoneum injects the monoclonal hybridoma strain 5 × 10 of aflatoxin B1 to Intraperitoneal injection sterilizing paraffin oil 0.5ml/
7individual/only, gather ascites after 7 days.Carry out ascites by sad-saturated ammonium sulfate method to purify ,-20 DEG C of preservations.
2. the preparation of polyclonal antibody
Adopt new zealand white rabbit as immune animal, with aflatoxin B1 haptens and bovine serum albumin(BSA) conjugate for immunogene, immunizing dose is 1.5mg/kg, when head exempts from, the Fu Shi of immunogene and equivalent is helped (originating the same) completely and be mixed and made into emulsifying agent, neck dorsal sc multi-point injection, interval is got same dose immunogene for 3 ~ 4 weeks and is added equivalent incomplete Freund's adjuvant (originating the same) mixing and emulsifying, and booster immunization is once, immunity 5 times, does not add adjuvant for the last time altogether.Take a blood sample after last immune 10 days, measure serum antibody titer, Culling heart blood, purifies by sad-saturated ammonium sulfate method and obtains polyclonal antibody.
3. the preparation process of sheep anti mouse antiantibody: using sheep as immune animal, carries out immunity for immunogene to pathogen-free domestic sheep with mouse source antibody, obtains sheep anti mouse antiantibody; The preparation of goat-anti rabbit antiantibody: using sheep as immune animal, carries out immunity for immunogene to pathogen-free domestic sheep with rabbit source antibody, obtains goat-anti rabbit antiantibody.
4. the preparation of ELISA Plate
Be buffered liquid with bag and 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 DEG C of incubation 2h or 4 DEG C spend the night, incline coating buffer, wash 2 times with the concentrated cleaning solution of dilution, each 30 seconds, get rid of liquid in hole, after absorbing residual moisture with thieving paper, 200 μ l confining liquids are added, 37 DEG C of incubation 2h, liquid in hole of inclining in every hole, with the vacuum seal of aluminium film after dry, 4 DEG C of dry places save backup.
The carbonate buffer solution of coating buffer for for pH value being 9.6,0.1mol/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 is w/v.
5. the preparation of enzyme labeling sheep anti mouse antiantibody
The Over-voltage protection after improveing is adopted to carry out coupling antiantibody and horseradish peroxidase (HR).Traditional Over-voltage protection requires that the molar concentration rate of enzyme and antiantibody in reflection system is 4: 1; Because horseradish peroxidase produces many sites be combined with antiantibody under the effect of Strong oxdiative, the horseradish peroxidase molecule of such activation act as the bridge connecting each molecule, reduce the enzymatic activity of enzyme marker, make to be mixed with many condensates in the conjugate of preparation, in order to address this problem, traditional method improves by we, that is:
1) amino closed process is eliminated, because it is seldom actual to produce self the amino amino connected.
2) horseradish peroxidase is reduced: the volumetric molar concentration ratio to 2 of antiantibody: 1, the method after 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 detecting aflatoxin B1, make it comprise following component:
(1) bag is by the ELISA Plate of aflatoxin B1 coupled antigen;
(2) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(3) aflatoxin B1 monoclonal antibody working fluid;
(4) aflatoxin B1 standard solution 6 bottles, 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) substrate nitrite ion is made up 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 pH value is 7.4, the phosphate buffer of 0.2mol/L containing 1.0% Tween-20,0.4 ‰ sodium azide, and described number percent is w/v.
(8) the concentrated liquid that redissolves is pH7.6, and containing 4% casein, the phosphate buffer of 0.15mol/L, described number percent is w/v.
The detection of aflatoxin B1 in embodiment 3 actual sample
Sample pre-treatments
(1) oil (peanut oil, blending stock, corn oil, soya-bean oil equal samples)
Take 2.0 ± 0.05g edible oil sample in 50ml polystyrene centrifuge tube; Add 4ml methanol-water solution, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 2.0ml supernatant liquor in 50ml polystyrene centrifuge tube, add 2.0ml deionized water, then add 6ml methenyl choloride, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Remove supernatant liquid, get 3ml lower floor organic phase in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, dissolve dried residue with vortex instrument whirling motion 30s, add 1ml redissolution liquid working fluid, with vortex instrument whirling motion 30s, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper strata normal hexane phase, takes off layer aqueous phase 50 μ l for analyzing.
(2) peanut (raw peanut, ripe peanut and salt peanut equal samples)
Take 2.0 ± 0.05g peanut sample in 50ml polystyrene centrifuge tube; Add 3.0ml acetonitrile, then add 3.0ml deionized water, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 3.0ml supernatant liquor in 50ml polystyrene centrifuge tube, add 4.5ml methenyl choloride, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Remove supernatant liquid, get 3ml lower floor organic phase in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, dissolve dried residue with vortex instrument whirling motion 30s, add 1ml redissolution liquid working fluid, with vortex instrument whirling motion 30s, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper strata normal hexane phase, takes off layer aqueous phase 50 μ l for analyzing.
(3) cereal (analysis for soybean powder, rice meal, corn flour, flour equal samples)
Take 2.0 ± 0.05g cereal sample in 50ml polystyrene centrifuge tube; Add 4.0ml acetonitrile, then add 2.0ml deionized water, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 3.0ml supernatant liquor in 50ml polystyrene centrifuge tube, add 6ml methenyl choloride, vibration 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Remove supernatant liquid, get 4ml lower floor organic phase in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, dissolve dried residue with vortex instrument whirling motion 30s, add 1ml redissolution liquid working fluid, with vortex instrument whirling motion 30s, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper strata normal hexane phase, takes off layer aqueous phase 50 μ l for analyzing.
2. detect with kit
Aflatoxin B1 standard solution or sample solution 50 μ l is added in the ELISA Plate micropore being coated with aflatoxin B1 coupled antigen, add ELIAS secondary antibody 50 μ l at random, add aflatoxin B1 monoclonal antibody working fluid 50 μ l again, with cover plate mould shrouding, 30min is reacted in 37 DEG C of constant temperature ovens, pour out liquid in hole, every hole adds 250 μ l cleansing solutions, liquid in hole is poured out after 30s, repeat operation and wash plate altogether 5 times, finally remove residual moisture with thieving paper, every hole adds substrate nitrite ion A liquid urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine (TMB), to vibrate gently mixing, 37 DEG C of constant temperature oven lucifuge colour developing 15min, every hole adds 2mol/L stop buffer hydrochloric acid 50 μ l, to vibrate gently mixing, 450nm place is set in microplate reader wavelength, measure every hole absorbance (OD value).
3. Analysis of test results
With the absorbance values (B) of the standard solution of the obtained each concentration absorbance (B divided by first standard solution (0 standard)
0) be multiplied by 100% again, obtain percentage absorbance.With the semilog value of aflatoxin B1 standard concentration (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map.By the percentage absorbance of same way calculation sample solution, the concentration of each sample corresponding, then can read the residual quantity of aflatoxin B1 from typical curve.
The mensuration of embodiment 4 kit quality
1 standard items precision test:
Respectively the ELISA Plate prepared from three different time periods respectively extract a collection of ELISA Plate out, often criticize each extraction 10 kits, 20 micropores extracted out by every plate, measure the absorbance of 0.2 μ g/L standard solution, calculate the coefficient of variation.
Table 2 standard repeatability test (CV%)
Can be drawn by above-mentioned test findings, often criticize each 10 the standard items coefficient of variation of kit between 2.4%-8.1%, meet the regulation that precision is less than or equal to 25%.
2 sample preci-sion and accuracy tests
A) sample precision test:
With the aflatoxin B1 of 0.2 μ g/L concentration, oil, peanut, cereal are carried out to interpolation and measures, get the kit of three different batches respectively, each concentration repeats 5 times and measures, and calculates the coefficient of variation respectively, the results are shown in Table 3.
This repeatability of table 3 oil sample is tested
The test of table 4 peanut sample repeatability
The test of table 5 cereal sample repeatability
Result shows that the coefficient of variation of oil, peanut, cereal sample is all between 7.3%-10.8%, met that " " Ministry of Agriculture's file " agriculture doctor sends out [2005] No. 17 annex 2 kits and puts on record with reference to the 4th precision standard in judgment criteria.
B) sample accuracy test
The aflatoxin B1 standard solution getting two concentration is respectively 0.2 μ g/kg, 0.4 μ g/kg, carries out interpolation recovery test respectively to sample, each concentration do 4 parallel, accuracy in computation.
The accuracy of table 6 kit
Result shows that oil, peanut, cereal sample TIANZHU XINGNAO Capsul are between 76.9%-87.9%.Meet " Ministry of Agriculture's file " agriculture doctor [2005] No. 17 annex 2 kits to put on record with reference to standard of accruacy in judgment criteria.
3 kits preserve experiment
Kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, aflatoxin B1 added practical measurement value all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 12 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 5 days, measurement result also shows that kit indices is completely normal.Can show that kit can preserve more than 12 months at 2 ~ 8 DEG C from above result.
Claims (7)
1. detect an enzyme linked immunological kit for aflatoxin B1, comprise coating antigen and enzyme marker, described coating antigen is selected from the conjugate of aflatoxin B1 haptens and carrier protein, aflatoxin b1 antibody and antiantibody, when described coating antigen is the conjugate of aflatoxin B1 haptens and carrier protein, described enzyme marker is enzyme mark antiantibody, when described coating antigen is aflatoxin b1 antibody, described enzyme marker is the conjugate of enzyme mark haptens and carrier protein, when described coating antigen is antiantibody, described enzyme marker is the conjugate of enzyme mark aflatoxin B1 haptens and carrier protein, described aflatoxin B1 haptens is by aflatoxin B1 and oxammonium hydrochloride condensation reaction, obtained by condensation reaction with succinic anhydride again, described aflatoxin b1 antibody is aflatoxin B1 monoclonal antibody, prepare using the conjugate of aflatoxin B1 haptens and carrier protein as immunogene, described carrier protein is ovalbumin, bovine serum albumin, mouse haemocyanin, thyroprotein, rabbit serum proteins, human albumin, hemocyanin or fibrinogen, described aflatoxin B1 monoclonal antibody is secreted by hybridoma cell strain D-2-2CGMCC No.5130 and is produced.
2. enzyme linked immunological kit according to claim 1, is characterized in that the marker enzyme of described enzyme marker is horseradish peroxidase or alkaline phosphatase.
3. enzyme linked immunological kit according to claim 1, is characterized in that described antiantibody is sheep anti mouse antiantibody.
4. enzyme linked immunological kit according to claim 1, 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 pH value is 7.2-7.5, containing 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, containing 2-4% casein, and the phosphate buffer of 0.1-0.2mol/L; Described substrate nitrite ion is made up 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; The carbonate buffer solution of described bag is buffered liquid to be pH value be 9.2-9.6,0.1-0.2mol/L; Described confining liquid is for containing 5-8% skimmed milk power, and the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described number percent is w/v.
5. enzyme linked immunological kit according to claim 2, it is characterized in that when marker enzyme is horseradish peroxidase, described developer is made up 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 is alkaline phosphatase, developer is nitro phosphate buffer, and stop buffer is 1 ~ 2mol/L sodium hydroxide solution.
6. detect a method for aflatoxin B1, comprise the following steps;
(1) sample pre-treatments;
(2) detect with the enzyme linked immunological kit described in any one of claim 1-5;
(3) testing result is analyzed.
7. preserving number is the aflatoxin B1 monoclonal antibody hybridoma cell strain D-2-2 of CGMCC No.5130.
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