CN101241131A - Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method - Google Patents

Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method Download PDF

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Publication number
CN101241131A
CN101241131A CN 200810020746 CN200810020746A CN101241131A CN 101241131 A CN101241131 A CN 101241131A CN 200810020746 CN200810020746 CN 200810020746 CN 200810020746 A CN200810020746 A CN 200810020746A CN 101241131 A CN101241131 A CN 101241131A
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China
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afb
ota
antibody
bsa
ochratoxin
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黄飚
马智鸿
张艺
朱岚
张珏
金坚
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Jiangsu Institute of Nuclear Medicine
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Jiangsu Institute of Nuclear Medicine
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Abstract

A reagent kit for detecting ochratoxin A and aflatoxin B1 at the same time and method thereof are provided, which belongs to fields of time-resolved fluoroimmunoassay. Micro plate is coated with OTA-BSA,AFB1-BSA, adding OTA, AFB1 standard or sample, and then adding OTA monoclonal antibody, AFB1 polyclonal antibody. Dissociated OTA, AFB1 and OTA-BSA, AFB1-BSA on micro plates competing corresponding antibody, and antibody which haven't be connected is eliminated after washing, adding Sm<3+>-goat anti rat and Eu<3+>- goat anti rabbit, labeled antibody haven't be connected after immune reaction are eliminated after washing. After adding enhancement solution, detecting Eu and scythe fluorescence intensity cps respectively which are inversely proportional to intensity of OTA and AFB1 in sample respectively by time resolved luminoscope, comparing standard curve and the content of OAT and AFB1 in sample is determined. The reagent kits of present invention has merits of simple structure, convenient operation and low cost, two examining results of OAT and AFB1 are obtained by one operation, the present invention is used for detecting of grain, feed and food.

Description

A kind of ochratoxin A and AFB of detecting simultaneously 1Kit and detection method thereof
Technical field
A kind of ochratoxin A and AFB of detecting simultaneously 1Kit and detection method thereof, belong to time resolved fluoro-immunoassay (TRFIA) technical field, be used for grain, feed and food ochratoxin A (OTA) and AFB 1(AFB 1) detection of content.
Background technology
Ochratoxin A (OTA) is the mycetogenetic a kind of toxin of fungi aspergillus ochraceous and several Penicillium.The kidney that OTA has been proved to be animal and people produces infringement, it also is a kind of carcinogen, the poisoning that mycotoxin causes is passed through mostly by the grain of mould contamination, oil crops and fermented food etc. cause, and the mycotoxin poisoning often shows as tangible region and seasonality, clinical manifestation is comparatively complicated, and acute poisoning, slow poisoning and carcinogenic, teratogenesis and mutagenesis etc. are arranged.OTA is all separable arriving in most of cereal, comprises barley, wheat, oat, corn, coffee bean etc., also can be polluted with the poultry of these cereal as feed.
The 56th the FAO/WHO food additives joint specialist council (JECFA) meeting has been carried out hazard assessment to OTA, the conclusion that draws is in the body and in vitro test shows that OTA has genetoxic, consider that cereal such as wheat, barley and rye are to take in the main food variety of OTA, the limit standard of OTA in these foods is formulated in decision, and meeting thinks in these foods and the goods thereof that it is 5 μ g/kg that OTA limits the quantity of.Up to the present, existing 11 countries have formulated the limit standard of the OTA in food (1-50 μ g/kg) and the feed (100-1000 μ g/kg).
AFB 1(AFB 1) be a group of supervirulent secondary metabolite, especially AFB that fungi aspergillus flavus Hspergillus flavus and aspergillus parasiticus A.parasilicus bacterial strain produce 1For strong polluter, have a very wide distribution, can cause the mankind, the acute poisoning death of all feeds animal, can also cause carcinogenic, teratogenesis, mutagenesis is even the content of tens μ g/kg still has very big toxicity.More than the aflatoxin 1mg/kg severe toxicity is arranged in food and the feed.Its toxicity is 10 times of potassium chloride, is 68 times of arsenic.After the edible aflatoxin-contaminated serious food heating can appear, stomachache, vomiting, anorexia, severe patient hepatosplenomegaly occurs in 2～3 weeks, hepatalgia, skin and mucosa xanthochromia, ascites, toxic hepatic disease such as edema of lower extremity and dysfunction of liver symptom also cardiac dilatation, pulmonary edema may occur, even spasm, diseases such as stupor.Owing to be difficult for preventing food by fungal contamination, so people pay special attention to the possibility of long-term edible low dosage aflatoxin contamination.Because aflatoxin is B particularly 1Be potential carcinogen, people contact the aflatoxin of low dosage for a long time may be influential, and international cancer research institution was with AFB in 1988 1Classify 1A class carcinogen as.
AFB 1Be the strongest carcinogen, its method threshold quantity in food is very low, and its limit standard is 2 μ g/kg.EU Committee replenishes the toxin limit standard in the infant food.Draft specifies, in comprising the infant food of cereal preparation, the AFB of toxicity maximum 1Maximum to limit the quantity of be 0.05 μ g/kg.
In view of AFB 1With the harm of OTA, be starved of set up highly sensitive, measurement range is wide, AFB conveniently 1Assay method with biotoxins such as OTA.Since in the security of cereal etc. detects, AFB 1With OTA often be the project that must detect simultaneously, in homogeneous operation, carry out high-sensitive OTA and AFB 1Synchronous detection can increase work efficiency, reduce application cost.
Present OTA and AFB 1Assay method have multiple, as thin-layered chromatography TLC, high performance liquid chromatography HPLC, immunofluorescence staining, but when having operating cost, instrument and equipment costliness, complicated operation and be not suitable for the shortcomings such as detection of batch samples.Immunoassay, highly sensitive, easy and simple to handle because its high specificity, do not need directly to contact toxin, and be particularly suitable for the advantage such as detection of batch samples and more and more paid attention to and adopt by people.However, the sensitivity that enzymoimmunoassay commonly used detects and the stability of reagent are also not satisfied, and can't carry out OTA and AFB simultaneously 1Measure.
Time resolved fluoro-immunoassay method (TRFIA) is the immunoassay of new development.TRFIA is the technology that the sensitivity of rare earth ion, characteristic such as stable are combined with immunoassay.The ultimate principle of its analysis is as follows: 1. make antigen or antibodies to certain surface of solid phase carriers, and keep its immunocompetence.2. make antigen or antibody and certain rare earth ion connect into labelled antigen or antibody, this labelled antigen or antibody had both kept its immunocompetence, kept the fluorescent characteristic of rare earth ion again.When measuring, reacted examining sample (mensuration antibody or antigen wherein) and labelled antigen or antibody antigen or antibody by different steps and surface of solid phase carriers.3. the method with washing makes the antigen antibody complex that forms on the solid phase carrier separate with other materials, and the amount that is combined in examined object matter in rare earth ion amount and the sample on the solid phase carrier at last becomes certain ratio.By the fluorescence of slack time measurement rare earth ion, eliminate the interference of natural fluorescence, thereby make assay method reach very high susceptibility.TRFIA also has a technical advantage, promptly can carry out many index analysis, if adopt different antigen or the antibody of rare earth ion mark such as europium, samarium, dysprosium, terbium simultaneously, because the emission light difference of rare earth ions such as europium, samarium, dysprosium, terbium just can obtain a plurality of data in once analyzing.
Summary of the invention
The object of the present invention is to provide a kind of ochratoxin A and AFB of detecting simultaneously 1Kit and detection method thereof, belong to time resolved fluoro-immunoassay (TRFIA) technical field, be used for grain, feed and food ochratoxin A (OTA) and AFB 1(AFB 1) detection of content.
Technical scheme of the present invention: this detects OTA and AFB 1Kit be by 1,96 or 48 holes bags by plate, 2, damping fluid, 3, contain ochratoxin A and AFB 1Standard, 4, the antibody dried frozen aquatic products of mouse-anti ochratoxin A, 5, rabbit aspergillus flavus resisting toxin B 1The antibody dried frozen aquatic products, 6, the sheep anti-mouse antibody of samarium mark, 7, the goat anti-rabbit antibody of europium mark, 8, cleansing solution and 9, strengthen liquid and form.
Bag wherein by solid phase antigen, is used 50mmol/L pH9.6Na by plate (1) bag 2CO 3-NaHCO 3Damping fluid with ochratoxin A-bovine serum albumin OTA-BSA, AFB 1-bovine serum albumin AFB 1-BSA is diluted to separately, and final concentration all is that 10mg/L is as coating buffer, 96 or 48 each hole of hole microwell plate add 100 μ L, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the above-mentioned damping fluid sealing that 150 μ L contain 3g/L BSA, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
Wherein contain ochratoxin A and AFB 1Standard (3), respectively from OTA, AFB 1Mix obtaining in the pure product after the dilution again, dilution is a methyl alcohol: water=3: 7, totally 6 bottles, the OTA concentration in each bottle is followed successively by: 0ng/mL, 0.1ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and AFB 1Concentration is followed successively by: 0ng/mL, 0.05ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL.
The antibody dried frozen aquatic products (4) of mouse-anti ochratoxin A wherein is the monoclonal antibody of the anti-ochratoxin A that obtained by ochratoxin A-hemocyanin (OTA-KLH) immune mouse.
Rabbit aspergillus flavus resisting toxin B wherein 1Antibody dried frozen aquatic products (5), for by AFB 1-hemocyanin (AFB 1-KLH) the aspergillus flavus resisting toxin B that obtains of immunizing rabbit 1Polyclonal antibody.
The sheep anti-mouse antibody (6) of samarium mark wherein with the sheep anti-mouse antibody of buying, to pH9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched sheep anti-mouse antibody and adds Sm 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
The goat anti-rabbit antibody (7) of europium mark wherein with the goat anti-rabbit antibody of buying, to pH9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched goat anti-rabbit antibody and adds Eu 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
Damping fluid wherein (2): contain 8mmol/L NaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1mL/L Tween-80 and 0.1%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8; Cleansing solution (8): 14.5mmol/L NaCl, 0.2mL/L Tween-80 and 0.2%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8; Strengthen liquid (9): 1L pH3.2 Potassium Hydrogen Phthalate damping fluid wherein contains 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1mL triton x-100.
The present invention mainly adopts time resolved fluoro-immunoassay method (TRFIA) to detect OTA and AFB 1Adopt TRFIA to detect OTA and AFB simultaneously 1Technology, mainly be Eu 3+Mark goat anti-rabbit antibody and Sm 3+The application of mark sheep anti-mouse antibody.
The basis of measuring is the labelled immune reaction.Microwell plate is coated with OTA-BSA, AFB jointly 1-BSA adds OTA, AFB 1Standard or sample add OTA monoclonal antibody, AFB again 1Polyclonal antibody.Free OTA, AFB 1With OTA-BSA and the AFB on the microwell plate 1-BSA competes corresponding antibody, does not have the OTA monoclonal antibody and the AFB that connect 1Polyclonal antibody is washed to be removed, and adds Sm 3+-sheep anti-mouse antibody and Eu 3+-goat anti-rabbit antibody, labelled immune reaction back does not have the Sm of connection 3+-sheep anti-mouse antibody and Eu 3+-goat anti-rabbit antibody is washed to be removed.After adding enhancing liquid, differentiate luminoscope with the time and measure europium and samarium fluorescence intensity cps respectively, the fluorescence intensity of samarium and the OTA concentration in the sample are inversely proportional to, the fluorescence intensity of europium and the AFB in the sample 1Concentration is inversely proportional to, and the reference standard curve can be determined OTA and AFB in the sample 1Content.
Detect ochratoxin A and AFB simultaneously with described kit 1Method, get and be coated with OTA-BSA and AFB 1The micropore bag of-BSA is added OTA, AFB by plate 1Standard or the sample handled well add anti-OTA, AFB again in micropore separately 1Antibody, oscillating reactions, cleansing solution washing, add the sheep anti-mouse antibody of samarium mark and the goat anti-rabbit antibody of europium mark, carry out the labelled immune reaction, the cleansing solution washing, add and strengthen the fluorescence intensity cps that measures europium and samarium after liquid vibrates respectively, OTA and AFB in the reference standard curve calculation sample 1Content.
Detect ochratoxin A and AFB simultaneously 1Method, concrete operations are: get be coated with OTA-BSA and AFB1-BSA the micropore bag by plate, add OTA, the AFB of 50 μ L 1Standard or the sample handled well add 25 μ L and make thinning agent with damping fluid (2) in micropore separately, and the OTA antibody of dilution in 1: 10 adds 25 μ L and makes thinning agent with damping fluid (2), the AFB of dilution in 1: 10 1Antibody, 25-37 ℃ vibrated 0.5-1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior; 50 μ L Eu of damping fluid (2) dilutions in 1: 10 in addition 3+-goat anti-rabbit antibody, in addition 50 μ L Sm of damping fluid (2) dilutions in 1: 10 3+-sheep anti-mouse antibody, 25-37 ℃ vibrated 0.5-1 hour, and washed six times with cleansing solution; Add the vibration of 200 μ L enhancing liquid and measure europium and samarium fluorescence intensity cps respectively, OTA from the typical curve calculation sample and AFB after 5 minutes 1Content.
Beneficial effect of the present invention: OTA and AFB are provided when providing 1Kit is simple in structure, and is easy to use, cheapness, and single job can obtain OTA and AFB simultaneously 1Two testing results.
Description of drawings
Fig. 1: detect ochratoxin A and AFB simultaneously 1The kit synoptic diagram.1,96 or 48 holes bags is by plate, and 2, damping fluid, 3, contain ochratoxin A and AFB 1Standard, 4, the antibody dried frozen aquatic products of mouse-anti ochratoxin A, 5, rabbit aspergillus flavus resisting toxin B 1The antibody dried frozen aquatic products, 6, the sheep anti-mouse antibody of samarium mark, 7, the goat anti-rabbit antibody of europium mark, 8, cleansing solution, 9, strengthen liquid.
Fig. 2: OTA-TRFIA canonical plotting.
Fig. 3: AFB 1-TRFIA canonical plotting.
Embodiment
Embodiment 1 preparation kit and detection corn sample
Eu 3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 1-2mL that is dissolved in 50mmol/L, pH7.0PBS, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/L Na that contains 0.155mol/L NaCl 2CO 3-NaHCO 3The pH8.5 damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis 280-0.74A 260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.The goat anti-rabbit antibody of getting after 500 μ L dilute adds the Eu that contains 0.2mg 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu 3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through Sepharose CL-6B post (1 * 40cm) chromatography, A with 80mmol/L, pH7.8 Tris-HCl damping fluid balance 280Protein peak is collected in monitoring, and the dilution packing is standby.
Sm 3+The preparation of-sheep anti-mouse antibody:
Conversion buffered condition is the same.The sheep anti-mouse antibody of getting after 500 μ L dilute adds the Sm that contains 0.2mg 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Sm 3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through Sepharose CL-6B post (1 * 40cm) chromatography, A with 80mmol/L, pH7.8 Tris-HCl damping fluid balance 280Protein peak is collected in monitoring, and the dilution packing is standby.
Bag is prepared by the plate solid phase antigen:
With 50mmol/L, pH9.6 Na 2CO 3-NaHCO 3Damping fluid with OTA-BSA, AFB 1-BSA is diluted to separately, and final concentration all is that 10mg/L is as coating buffer, 96 each hole of hole microwell plate add 100 μ L, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the above-mentioned damping fluid sealing that 150 μ L contain 3g/L BSA, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent:
(1) contains ochratoxin A and AFB 1Standard: respectively from OTA, AFB 1Mix obtaining in the pure product after the dilution again, dilution is a methyl alcohol: water=3: 7, totally 6 bottles, the OTA concentration in every bottle is followed successively by: 0ng/mL, 0.1ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and AFB 1Concentration is followed successively by: 0ng/mL, 0.05ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL.
(2) damping fluid: contain 8mmol/L NaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1mL/L Tween-80 and 0.1%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8.
(3) cleansing solution: 14.5mmol/L NaCl, 0.2mL/L Tween-80 and 0.2%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8.
(4) strengthen the preparation of liquid: 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid wherein contains 15 μ mol β-naphthoyltrifluoroacetones (β-NTA), 50 μ mol trioctyl-phosphine oxide (TOPO), 1mL triton x-100 (Triton X-100).
The reagent that kit provides:
Reagent in each box enough carries out 96 measurements, and the material in the box is as follows:
(1) .1 * 96 orifice plates (8 * 12 hole can be split as single hole) are coated with OTA-BSA, AFB 1-BSA.
(2) .1 * damping fluid: 30mL.
(3) .6 * OTA, AFB 1Titer, the 1.0mL/ bottle, the OTA concentration in every bottle is followed successively by: 0ng/mL, 0.1ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and AFB 1Concentration is followed successively by: 0ng/mL, 0.05ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL.
(4) .1 * OTA antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5) .1 * AFB 1The antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(6) .1 * Sm 3+-sheep anti-mouse antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(7) .1 * Eu 3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(8) .1 * cleansing solution: 30mL, the time spent is with distilled water dilution in 1: 25.
(9) .1 * enhancing liquid: 20mL.
The reagent that the laboratory should be provided for oneself:
Methyl alcohol.
70% methanol solution: the pure methyl alcohol of 30mL distilled water or deionized water and 70mL is mixed with 70% methanol solution.
Distilled water or deionized water.
Points for attention before measuring
1, uses before all reagent to be gone up to room temperature (18-30 ℃).
2, immediately all reagent are put back to 2-8 ℃ of preservation after the use.
If the hyperchannel pipettor is used in the big suggestion of 3 sample sizes.
4, hatch in the process at all constant temperature, avoid irradiate light, use the cap covers micropore.
5, taking-up needs microwell plate and the framework with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2-8 ℃.
Concrete detection step is as follows:
Just corn sample is crushed to 20 orders earlier, gets 5 gram samples and is placed in the test tube, adds extract 12.5mL (methyl alcohol: water=7: 3).Jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua.Get 1mL filtrate and dilute with 1mL distilled water or deionized water, standby.
Get and be coated with OTA-BSA and AFB 1The micropore bag of-BSA is added OTA, the AFB of 50 μ L by plate 1Standard or the sample handled well add 25 μ L and make thinning agent with damping fluid (2) in micropore separately, and 1: 10 dilution OTA antibody adds 25 μ L and makes thinning agent with damping fluid (2), 1: 10 dilution AFB 1Antibody, 25-37 ℃ vibrated 0.5-1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior; 50 μ L Eu of damping fluid (2) dilutions in 1: 10 in addition 3+-goat anti-rabbit antibody, in addition 50 μ L Sm of damping fluid (2) dilutions in 1: 10 3+-sheep anti-mouse antibody, 25-37 ℃ vibrated 0.5-1 hour, and washed six times with cleansing solution; Add the vibration of 200 μ L enhancing liquid and measure europium and samarium fluorescence intensity cps respectively, OTA from the typical curve calculation sample and AFB after 5 minutes 1Content sees Table 1.1,1.2 and Fig. 2 .1,2.2, and the sample OTA concentration that this example is extracted is 0.21ng/mL, AFB 1Concentration is 0.35ng/mL (corn sample OTA, AFB 1Content is respectively 1.05 μ g/kg, 1.75 μ g/kg).
Table 1.1
The OTA standard point
OTA concentration (ng/mL) 0 0.1 1 5 10 50 Corn sample
Sm 3+Fluorescent value (cps) 37621 35233 26811 14053 8225 2312 32367
Table 1.2
AFB 1Standard point
AFB 1Concentration (ng/mL) 0 0.05 0.5 1 10 100 Corn sample
Eu 3+Fluorescent value (cps) 406914 372062 143016 93305 35102 22351 174817
Embodiment 2 preparation kits
Bag is prepared by the plate solid phase antigen: with 50mmol/L, pH9.6 Na 2CO 3-NaHCO 3Damping fluid with OTA-BSA, AFB 1-BSA be diluted to separately final concentration all be 10mg/L as coating buffer, 48 each hole of hole microwell plate add 100 μ L, 4 ℃ of placements are spent the night, and discard coating buffer, wash three times; Add the above-mentioned damping fluid sealing that 150 μ L contain 3g/L BSA, 4 ℃ of placements are spent the night, and discard confining liquid, and vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent: with embodiment 1.
The reagent that kit provides:
Reagent in each box enough carries out 48 measurements, and the material in the box is as follows:
(1) .1 * 48 orifice plates (4 * 12 hole can be split as single hole) are coated with OTA-BSA, AFB 1-BSA.
(2) .1 * damping fluid: 30mL.
(3) .6 * OTA, AFB 1Titer, the 1.0mL/ bottle, totally 6 bottles, the OTA concentration in every bottle is followed successively by: 0ng/mL, 0.1ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and AFB 1Concentration is followed successively by: 0ng/mL, 0.05ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL.
(4) .1 * OTA antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5) .1 * AFB 1The antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(6) .1 * Sm 3+-sheep anti-mouse antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(7) .1 * Eu 3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(8) .1 * cleansing solution: 30mL, the time spent is with distilled water dilution in 1: 25.
(9) .1 * enhancing liquid: 20mL.
The reagent that the laboratory should be provided for oneself is identical with embodiment 1.Points for attention are with embodiment 1 before measuring.
The concrete step that detects is with embodiment 1.
Embodiment 3
The reagent that kit provides is identical with embodiment 1, is used to detect wheat samples.
Concrete detection step is as follows:
Earlier wheat samples is handled: wheat samples is crushed to 20 orders, gets 5 gram samples and be placed in the test tube, add extract 12.5mL (methyl alcohol: water=7: 3).Jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua.Get 1mL filtrate and dilute with 1mL distilled water or deionized water, standby.
Get and be coated with OTA-BSA and AFB 1The micropore bag of-BSA is added OTA, the AFB of 50 μ L by plate 1Standard or the sample handled well add 25 μ L and make thinning agent with damping fluid (2) in micropore separately, and 1: 10 dilution OTA antibody adds 25 μ L and makes thinning agent with damping fluid (2), 1: 10 dilution AFB 1Antibody, 25-37 ℃ vibrated 0.5-1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior; 50 μ L Eu of damping fluid (2) dilutions in 1: 10 in addition 3+-goat anti-rabbit antibody, in addition 50 μ L Sm of damping fluid (2) dilutions in 1: 10 3+-sheep anti-mouse antibody, 25-37 ℃ vibrated 0.5-1 hour, and washed six times with cleansing solution; Add the vibration of 200 μ L enhancing liquid and measure europium and samarium fluorescence intensity cps respectively, OTA from the typical curve calculation sample and AFB after 5 minutes 1Content sees Table 2.1,2.2 and Fig. 2 .1,2.2, and the sample OTA concentration that this example is extracted is 0.17ng/mL, AFB 1Concentration is 0.08ng/mL (wheat samples OTA, AFB 1Content is respectively 0.85 μ g/kg, 0.4 μ g/kg).
Table 2.1
The OTA standard point
OTA concentration (ng/mL) 0 0.1 1 5 10 50 Wheat samples
Sm 3+Fluorescent value (cps) 37621 35233 26811 14053 8225 2312 33335
Table 2.2
AFB 1Standard point
AFB 1Concentration (ng/mL) 0 0.05 0.5 1 10 100 Wheat samples
Eu 3+Fluorescent value (cps) 406914 372062 143016 93305 35102 22351 327500

Claims (10)

1, a kind of ochratoxin A and AFB of detecting simultaneously 1Time resolved fluoro-immunoassay method kit, it is characterized in that by 96 or 48 holes bags by plate (1), damping fluid (2) contains ochratoxin A and AFB 1Standard (3), the antibody dried frozen aquatic products (4) of mouse-anti ochratoxin A, rabbit aspergillus flavus resisting toxin B 1Antibody dried frozen aquatic products (5), the sheep anti-mouse antibody of samarium mark (6), the goat anti-rabbit antibody of europium mark (7), cleansing solution (8) and strengthen liquid (9) and form.
2, kit according to claim 1, bag wherein by solid phase antigen, is used 50mmol/LpH9.6Na by plate (1) bag 2CO 3-NaHCO 3Damping fluid with ochratoxin A-bovine serum albumin OTA-BSA, AFB 1-bovine serum albumin AFB 1-BSA is diluted to separately, and final concentration all is that 10mg/L is as coating buffer, 96 or 48 each hole of hole microwell plate add 100 μ L, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the above-mentioned damping fluid sealing that 150 μ L contain 3g/LBSA, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
3, kit according to claim 1, wherein contain ochratoxin A and AFB 1Standard (3), respectively from OTA, AFB 1Mix obtaining in the pure product after the dilution again, dilution is a methyl alcohol: water=3: 7, totally 6 bottles, the OTA concentration in every bottle is followed successively by: 0ng/mL, 0.1ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and AFB 1Concentration is followed successively by: 0ng/mL, 0.05ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 100ng/mL.
4, kit according to claim 1, the antibody dried frozen aquatic products (4) of mouse-anti ochratoxin A wherein is the monoclonal antibody by ochratoxin A-anti-ochratoxin A that hemocyanin OTA-KLH immune mouse obtains.
5, kit according to claim 1, rabbit aspergillus flavus resisting toxin B wherein 1Antibody dried frozen aquatic products (5), for by AFB 1-hemocyanin AFB 1The aspergillus flavus resisting toxin B that-KLH immunizing rabbit obtains 1Polyclonal antibody.
6, kit according to claim 1, the sheep anti-mouse antibody (6) of samarium mark wherein with the sheep anti-mouse antibody of buying, to pH9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched sheep anti-mouse antibody and adds Sm 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
7, kit according to claim 1, the goat anti-rabbit antibody (7) of europium mark wherein with the goat anti-rabbit antibody of buying, to pH9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched goat anti-rabbit antibody and adds Eu 3+-N 2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
8, kit according to claim 1, damping fluid wherein (2): contain 8mmol/L NaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1mL/L Tween-80 and 0.1%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8; Cleansing solution (8): 14.5mmol/L NaCl, 0.2mL/LTween-80 and 0.2%NaN 3The Tris-HCl solution of 50mmol/L, pH7.8; Strengthen liquid (9): 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid wherein contains 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1mL triton x-100.
9, a kind ofly detect ochratoxin A and AFB simultaneously with the described kit of claim 1 1Method, it is characterized in that getting and be coated with OTA-BSA and AFB 1The micropore bag of-BSA is added OTA, AFB by plate 1Standard or the sample handled well add anti-OTA, AFB again in micropore separately 1Antibody, oscillating reactions, cleansing solution washing, add the sheep anti-mouse antibody of samarium mark and the goat anti-rabbit antibody of europium mark, carry out the labelled immune reaction, the cleansing solution washing, add and strengthen the fluorescence intensity cps that measures europium and samarium after liquid vibrates respectively, OTA and AFB in the reference standard curve calculation sample 1Content.
10, ochratoxin A and the AFB of detecting simultaneously according to claim 9 1Method, it is characterized in that: get and be coated with OTA-BSA and AFB 1The micropore bag of-BSA is added OTA, the AFB of 50 μ L by plate 1Standard or the sample handled well add 25 μ L and make thinning agent with damping fluid (2) in micropore separately, and 1: 10 dilution OTA antibody adds 25 μ L and makes thinning agent with damping fluid (2), 1: 10 dilution AFB 1Antibody, 25-37 ℃ of vibration 0.5-1 hour, cleansing solution is given a baby a bath on the third day after its birth time, in addition 50 μ L Eu of damping fluid (2) dilutions in 1: 10 3+-goat anti-rabbit antibody, in addition 50 μ L Sm of damping fluid (2) dilutions in 1: 10 3+-sheep anti-mouse antibody, 25-37 ℃ vibrated 0.5-1 hour, washed six times with cleansing solution, added the vibration of 200 μ L enhancing liquid and measured samarium and europium fluorescence intensity cps respectively, OTA from the typical curve calculation sample and AFB after 5 minutes 1Content.
CN 200810020746 2008-02-21 2008-02-21 Reagent kit for simultaneously detecting ochratoxin A and aspergillus flavus toxin B1 and its detection method Pending CN101241131A (en)

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CN102220286A (en) * 2010-12-21 2011-10-19 中国农业科学院油料作物研究所 Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof
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CN111781384A (en) * 2020-07-24 2020-10-16 浙江理工大学绍兴生物医药研究院有限公司 Immunoassay kit for detecting IgG and IgG4 of M-type phospholipase A2 receptors and detection method thereof
CN111781383A (en) * 2020-07-24 2020-10-16 浙江理工大学绍兴生物医药研究院有限公司 Immunoassay kit for detecting IgG (immunoglobulin G) of M-type phospholipase A2 receptor and detection method thereof
CN111909028A (en) * 2020-08-24 2020-11-10 北京石油化工学院 Preparation method of Eu/Tb (BTC) for detecting AFB1
CN111909028B (en) * 2020-08-24 2022-11-29 北京石油化工学院 Preparation method of Eu/Tb (BTC) for detecting AFB1
CN113341147A (en) * 2021-05-08 2021-09-03 郑州大学 Construction and application of dual-fluorescence immunoassay system for zearalenone and ochratoxin A

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