CN103308687A - Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut - Google Patents
Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut Download PDFInfo
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- CN103308687A CN103308687A CN 201210055729 CN201210055729A CN103308687A CN 103308687 A CN103308687 A CN 103308687A CN 201210055729 CN201210055729 CN 201210055729 CN 201210055729 A CN201210055729 A CN 201210055729A CN 103308687 A CN103308687 A CN 103308687A
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Abstract
The invention discloses a suspension chip detection method for simultaneously detecting various mycotoxins (aflatoxin B1, vomitoxin, T-2 toxin, and zearalenone). Complete antigens of the mycotoxins and carboxylated microspheres with different codes are coupled through an EDC method, and covalent binding is allowed. With a competition method, a suspension chip technology research is carried out. During detection, the conjugate and monoclonal antibodies of the mycotoxins are oscillated; standard products are added, such that corresponding small molecules of a detection target and the complete antigens compete for the limited monoclonal antibodies; centrifugation is carried out, and supernatant is removed; a biotinylated secondary antibody is added; centrifugation is carried out, and supernatant is removed; SA-PE is added, such that a composition of detection target antigen-monoclonal antibody-secondary antibody-SA-PE is obtained; a median fluorescence value is reduced with the increasing of the concentration of the small molecules, such that a multichannel competition standard curve is drawn. Methanol/water with a certain ratio is used for pre-treating peanut and corn, and multichannel standard curves in corn and peanut are respectively drawn with a negative treatment liquid as a diluting liquid. Through the researches on matrix effect, method accuracy, and method repeatability, various mycotoxins in corn and peanut can be simultaneously detected with the suspension chip method. With the process, a sensitivity reaches a pg-ng level, the result is stable and reliable, sample dose is low, and the method is simple and fast. According to the invention, a novel method is provided for the detection of various mycotoxins.
Description
Technical field
The invention provides and a kind ofly can detect four kinds of mycotoxins suspension chip method of (comprising AFB l, vomitoxin, T-2 toxin and zearalenone) simultaneously, particularly relate to a kind of suspending chip detection technique that detects multiple mycotoxin in corn and the peanut simultaneously.
Background technology
Fungi (Fungi) is that a class has cell membrane, does not contain chlorophyll, and the unrooted base of leaf is survived in saprophytic or parasitic mode, can carry out sexual or vegetative microorganism.Mycotoxin (Mycotoxin) also claim mycotoxin, is some mycetogenetic metabolic product or secondary metabolism product, and at present known just have a different mycotoxin of kind more than 350.Usually, mycotoxin pollutes cereal crops and animal feed, after entering biologic chain, can cause not only that agricultural product are mould to lose the loss of rotten, nutritional labeling and product quality reduces, even have serious harms such as carcinogenic, teratogenesis and mutagenesis, and also have characteristics such as hepatotoxicity, nephrotoxicity, reproduction disorder and immunosupress.This shows that mycotoxin can constitute a serious threat to food security and human health.Now, along with the great attention of the mankind to healthy and food security, the research of mycotoxin is also seemed more and more urgently with important.
Up to now, research to this piece both at home and abroad mainly concentrates in agricultural product and the feed on tens kinds of bigger mycotoxins of mankind's harm, (mainly be AFB l and Ml comprising aflatoxin, be Aflatoxin B1 and Aflatoxin M1, abbreviate AFB1 and AFM1 as), vomitoxin (has another name called deoxynivalenol, be Deoxynivalenol, DON), T-2 toxin (T-2toxin, T-2), zearalenone (has another name called the F-2 toxin, be Zearalenone, ZEN), ochratoxin A (Ochratoxin A, OTA), volt horse verticillium toxin (Fumonisin, F), patulin (Patulin, PTL), the series connection fusarium toxin (Moniliformin, MF), notalin (Citrinin, CIT), toxin (the Trichothecenes of trichothecene family, TS) etc., they generally have characteristics such as strong toxicity and pollution frequency height simultaneously.Accordingly, the target that the present invention studies is four kinds of mycotoxins, i.e. AFB1, DON, T-2 and ZEN.
At present, detecting mycotoxin method commonly used both at home and abroad mainly is high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA), though the former detection sensitivity height, sample pre-treatments is loaded down with trivial details, complicated operation, length consuming time, and equipment needed thereby is costliness also; The latter is easy and simple to handle, quick, but can only finish a kind of detection of mycotoxin at every turn, and for the detection of multiple toxin time-consuming, effort not only, workload is also bigger.
Suspending chip technology (Suspension array technology, SAT) be the multi-functional liquid phase chip analysis platform of U.S. Luminex company development in 1997, because reactions all in the liquid-phase chip all is that microsphere surface in being suspended in liquid phase environment is gained the name, claim liquid-phase chip (Liquid array/chip) again, full name is the synchronous analysis system of multi-functional many indexs (Flexible Multiple Analyte Profiling, xMAP) or the multifunctional suspending dot matrix (Multi-Analyte Suspension Array, MASA).This technology is unique new bio chip product that is used for clinical diagnosis by U.S. food Drug Administration (FDA) approval, technology such as set laser technology, micro-fluidic technologies, fast signal processing and data analysis system have been widely used in fields such as biomedicine, food security in one.
The detection principle of suspending chip is based on the polystyrene microsphere that a kind of diameter is 5.6 μ m, have two kinds of fluorescent materials on the microballoon, difference according to these two kinds of fluorescent material concentration proportionings, microballoon can be divided into 100 kinds (the up-to-date technology of Luminex company is by three kinds of different fluorescent materials being carried out proportioning, microballoon can being divided into 500 kinds).Can the different group molecule of coupling on the surface of microballoon, for example carboxyl, hydroxyl, biotin, Streptavidin etc. are to be adapted to the coupling to different probe molecule and microballoon.After the microballoon coupling of different probe molecules and corresponding numbering, these microballoons are blended in the reaction tube, add sample to be checked, detect when just can realize different target molecule.After reaction is finished, add the two anti-or corresponding materials that have the report fluorescence molecule, go up machine testing then.Thereby the suspending chip instrument is by the numbering qualitatively analyze target molecule to be checked of the multiple microballoon of identification, thereby the report fluorescence that is combined on the microballoon by identification then realizes analyzing quantitatively multiple target.The present invention also with the corn in the cereal crops, be representative with the peanut in the oil crops, realized that the using suspending chip method detects the purpose of four kinds of mycotoxins simultaneously.
The suspending chip technology of four kinds of mycotoxins of a kind of synchronous detection that the present invention studies, have high flux, multifunctionality, easy and simple to handle, good reproducibility, highly sensitive, accuracy advantages of higher, be applicable to the fast quantification of the little molecular contaminants of mycotoxin is detected, have broad application prospects at the detection range of environment and food security poisonous and harmful substance.
Summary of the invention
The objective of the invention is the defective at existing detection method existence, a kind of suspending chip detection technique of four kinds of mycotoxins fast and accurately simple to operate, sensitive is provided.
Another object of the present invention provides the suspending chip detection technique of four kinds of mycotoxins of a kind of synchronous detection, and based on this set up in corn and peanut simultaneously to the quantitative detecting method of four kinds of mycotoxins.
Technical scheme of the present invention is summarized as follows:
(1) four kind of mycotoxin comlete antigen respectively with the coupling of different coding microballoon;
Determining of (2) four kinds of mycotoxin monoclonal antibodies and biotinylation two anti-best effort concentration, and specific checking between the antigen-antibody;
The drafting of hyperchannel typical curve when (3) detecting four kinds of mycotoxins simultaneously;
(4) detect the foundation of four kinds of mycotoxin suspension chip methods in corn and the peanut simultaneously;
(5) the suspending chip method detects accuracy and the repetitive research of corn, peanut.
The coupling of preferred (1) four kind of mycotoxin comlete antigen of step and different coding microballoon is: with reference to the coupling method of the albumen that provides in the Luminex official website and carboxylated microballoon, the comlete antigen of four kinds of mycotoxins is carried out coupling by the carboxylated microballoon after certain working concentration and the activation, make in the subsequent experimental reacting hole contained microballoon＞2000/hole, be the best effort concentration of determining in the following step experiment that four kinds of mycotoxin monoclonal antibodies and biotinylation two are anti-, and the specificity between the checking antigen-antibody is prepared.
Determining of the best effort concentration that (2) four kinds of mycotoxin monoclonal antibodies of described step and biotinylation two resist, and specific being verified as between the antigen-antibody: prepare four kinds of mycotoxin monoclonal antibody (AFT-Ab earlier, DON-Ab, T-2-Ab and ZEN-Ab) solution of mixed liquor and biotinylation goat-anti mouse two anti-(Sec-Ab), each four concentration, after its equity mixing, the conjugate that adds four kinds of comlete antigens and microballoon, be accompanied with blank and negative control, in 37 ℃ of concussion 20-40min, carry out immune response, the centrifugal supernatant of abandoning afterwards, add SA-PE in 37 ℃ of concussion 20-40min, be transferred at last and go up machine testing in 96 orifice plates.Thereby obtain the best effort concentration of four kinds of mycotoxin monoclonal antibodies and Sec-Ab, and the specificity of four kinds of mycotoxin antigen-antibodies is analyzed, the drafting of hyperchannel typical curve is prepared when detecting four kinds of mycotoxins for going on foot down simultaneously in the experiment.
Being plotted as of hyperchannel typical curve when described step (3) detects four kinds of mycotoxins simultaneously: the best effort concentration mixed liquor of four kinds of mycotoxin monoclonal antibodies of preparation earlier, add the comlete antigen of each toxin and the vibration 5-15min of conjugate elder generation of microballoon again, add each little molecular solution vibration 15-25min again with the immune response that is at war with, and be provided with blank, negative control and positive control, the centrifugal supernatant of abandoning then, add certain density Sec-Ab solution vibration 10-20min afterwards, centrifugal abandoning adds SA-PE vibration 20-40min behind the supernatant again, is transferred at last and goes up machine testing in 96 orifice plates.Thereby detected the typical curve of four kinds of mycotoxins simultaneously, prepared for going on foot the foundation that detects four kinds of mycotoxin suspension chip methods in corn and the peanut in the experiment simultaneously down.
Described step (4) detects being established as of four kinds of mycotoxin suspension chip methods in corn and the peanut simultaneously:
Be extract carries out actual sample to corn and peanut pre-treatment with a certain proportion of methanol, and by suspending chip method four kinds of mycotoxin competition test curves of detection when drawing as matrix with the negative sample liquid of corn and the negative sample liquid of peanut respectively, analyze by the influence to matrix, judge whether this method of suspending chip can quantitatively detect four kinds of mycotoxins in corn and the peanut sample simultaneously.
Accuracy and repetitive research that described step (5) suspending chip method detects corn, peanut are: replace antibody diluent with negative corn-like liquid and negative peanut sample liquid, reclaim experimental identification suspending chip method by the mark-on to corn and peanut sample and detect accuracy and the repeatability of corn, peanut, thereby differentiate the quantitative detection whether this method can be applicable to four kinds of mycotoxins in corn and the peanut.
By a series of optimization experiment, the present invention can quantitatively detect the mark-on content of four kinds of mycotoxins in corn and the peanut easy, effectively, and sensitivity can reach pg～ng level level, is 3～4 orders of magnitude between detection zone, and it is less that result and actual value are compared deviation.
Description of drawings
Fig. 1 for suspending chip detect in detection synoptic diagram after AFT-BSA and 22#, DON-BSA and 26#, T-2-BSA and 30#, ZEN-BSA and the 32# coupling.
Fig. 2 is the chessboard optimization result of four kinds of mycotoxin monoclonal antibodies and biotinylation goat-anti mouse two anti-(Sec-Ab) when carrying out immune response simultaneously.
MFI value among the figure is MFI value (positive hole) and deducts MFI (blank), and the MFI value among the present invention all for this reason; Con. represents concentration among the figure.Wherein, 2-1 is the chessboard optimization result of AFT-Ab and Sec-Ab working concentration, 2-2 is the chessboard optimization result of DON-Ab and Sec-Ab working concentration, and 2-3 is the chessboard optimization result of T-2-Ab and Sec-Ab working concentration, and 2-4 is the chessboard optimization result of ZEN-Ab and Sec-Ab working concentration.
Fig. 3 is the specific detection result of four kinds of mycotoxin antigen-antibodies.
Fig. 4 detects the drafting of four kinds of mycotoxin typical curves simultaneously for suspending chip method in the different substrates.
Wherein, 4-1 is in antibody diluent, detects the competition curve figure of four kinds of mycotoxins simultaneously with the suspending chip method; 4-2 is in the negative solution of the corn after 20% methanol is handled, and detects the competition curve figure of four kinds of mycotoxins simultaneously with the suspending chip method; 4-3 is in the negative solution of the peanut after 40% methanol is handled, and detects the competition curve figure of four kinds of mycotoxins simultaneously with the suspending chip method.The three all is the competition test curves that use Origin 8.0 softwares to draw out.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is further elaborated.Following embodiment is for the ease of understanding the present invention better, but does not limit the present invention.
Experiment material required for the present invention is as follows:
Aflatoxin B1 (Aflatoxin B1, AFB1), vomitoxin (Deoxynivalenol, DON), T-2 toxin (T-2 Toxin, T-2), zearalenone (Zearalenone, ZEN): Israel Fermentek Ltd., total aflatoxin content comlete antigen (AFT-BSA), vomitoxin comlete antigen (DON-BSA), T-2 toxin comlete antigen (T-2-BSA), zearalenone comlete antigen (ZEN-BSA), aspergillus flavus resisting toxin total amount monoclonal antibody (AFT-Ab), emesis toxin monoclone antibody (DON-Ab), anti-T-2 toxin monoclone antibody (T-2-Ab), anti-zearalenone monoclonal antibody (ZEN-Ab): Huaan, Beijing Mai Ke Bioisystech Co., Ltd, blank carboxylated fluorescent microsphere (is numbered 22#, 26#, 30# and 32#): U.S. Luminex company, biotinylation goat anti-mouse igg (H+L), abbreviate Sec-Ab as: Wuhan Boster Biological Technology Co., Ltd., 1-ethyl-3-(3-dimethyl propyl) carbodiimide hydrochloride (EDC), the N-hydroxy-succinamide of sulfonation (S-NHS): U.S. Pierce company, the phycoerythrin of streptavidin (SA-PE): American I nvitrogen company, Tween-20, bovine serum albumin(BSA) (BSA): U.S. Sigma company.It is pure that other chemical reagent is homemade analysis, and used pure water is the Milli-Q ultrapure water.
Required key instrument is as follows:
Luminex 100 suspending chip systems are U.S. Luminex company, and MS3 digital display oscillator is German IKA company, and TGL-16C type high speed tabletop centrifuge is Anting Scientific Instrument Factory, Shanghai, and used aluminium-foil paper is that homemade Beijing is through company of HTC of section.
Being formulated as follows of main solution:
1 four kinds of mycotoxin comlete antigens of embodiment respectively with the coupling of different coding microballoon
The carboxylated microballoon of 22#, 26#, 30# and 32# that the present invention adopts Luminex company coding respectively with AFT-BSA, DON-BSA, four kinds of comlete antigen couplings of T-2-BSA, ZEN-BSA.
1.1 the activation of carboxylated microballoon
The brown vial that microballoon will be housed before using the kit coupling places under the room temperature places 30min.Use Aluminium Foil Package to be overlying on outside the pipe in the experiment process, it is fast that application of sample is wanted, and guarantees that indoor light is too not bright, prevents that as far as possible microballoon is exposed under the light for a long time, to reduce the probability of exposure.Before the activation, EDC and S-NHS are taken out from-20 ℃ of refrigerators, packing in a small amount, each time spent is respectively taken out a pipe, puts into the dry 1h of exsiccator under the room temperature in advance.If experiment does not use up as yet, can place-70 ℃ of superfreeze, use once again, otherwise discard.
(1) selects to have the microballoon of carboxyl as protein cross reaction, middling speed vortex oscillation microballoon 30s, ultrasonic 30s in water-bath then.
(2) 1 * cross-linking reaction in, sucking-off 100 μ L (1.25 * 10 from single microsphere suspension liquid that disperses to have carboxyl
6Beads/mL) in an Eppendorf pipe, react, with 14,000 * g this is managed centrifugal 4min, abandoning supernatant carefully.
(3) add 100 μ L microballoon cleaning buffer solutions, vortex oscillation 10s, ultrasonic 10s manages centrifugal 4min, abandoning supernatant carefully with 14,000 * g to this then.
(4) activate the granular substance of the resuspended microballoon of damping fluid with 80 μ L microballoons, vortex oscillation 30s, ultrasonic 30s in the water-bath.
(5) newly prepare EDC and S-NHS (respectively being 50mg/mL), after using microballoon activation damping fluid, with its adding.Add S-NHS and EDC (each 10 μ L) successively rapidly, vortex oscillation 30s coats the cross-linking reaction test tube and at room temperature uses vortex oscillation device middling speed vibration microballoon 20min with aluminium-foil paper.
(6) add the PBS of 150 μ L pH 7.4 again, with the microballoon 10s of high speed vortex oscillation activation.The microballoon 4min of the centrifugal activation of 14,000 * g, abandoning supernatant carefully then.Repeat this step 2 time.
1.2 the coupling of carboxylated microballoon and protein example
(1) in the microballoon of activation, adds protein example (5-12 μ g), with PBS adjustment final concentration to the 500 μ L of pH 7.4.Be overlying on centrifuge tube with Aluminium Foil Package and stir microballoon 2-4h with stirrer outward and at room temperature, perhaps stir down at 4 ℃ and spend the night.
(2) with the speed of 14,000 * g this is managed centrifugal 4min, abandoning supernatant carefully.
(3) clean microballoon with the PBS of 500 μ LpH 7.4, the centrifugal 4min of 14000 * g, abandoning supernatant carefully is not ultrasonic then.
(4) with the resuspended crosslinked microsphere of the confining liquid of 250 μ L, with medium speed's vortex oscillation 15s, coat centrifuge tube and at room temperature stir microballoon 20min with stirrer with aluminium-foil paper.
The centrifugal 4min of (5) 14000 * g, abandoning supernatant carefully.
(6) with 500 μ L deposit buffer solution for cleaning crosslinked microsphere, the centrifugal 6min of 16000 * g, abandoning supernatant carefully.
(7) with the resuspended crosslinked microsphere of 150 μ L deposit damping fluid, aluminium-foil paper coats the crosslinked microsphere pipe, and is standby in 4 ℃.
1.3 bag is by the counting of microballoon
Draw an amount of microballoon, after the dilution, determine the quantity of microballoon with haemocytometer or suspending chip instrument, and guarantee that every kind of contained microballoon number average of each reacting hole in the subsequent experimental is greater than 2000.The conjugate distribution plan of four kinds of comlete antigens and microballoon is seen accompanying drawing 1.
Determining of 2 four kinds of mycotoxin monoclonal antibodies of embodiment and biotinylation two anti-best effort concentration, and specific checking between the antigen-antibody
Utilize coupling to have the microballoon of comlete antigen to determine the best effort concentration that four kinds of mycotoxin monoclonal antibodies to be detected and biotinylation two are anti-, concrete steps are as follows:
(1) (concentration is followed successively by 25 to prepare four kinds of mycotoxin monoclonal antibody: AFT-Ab earlier, 50,100 and 200ng/mL), (concentration is followed successively by 2.5 to DON-Ab, 5,10 and 20ng/mL), (concentration is followed successively by 6.25 to T-2-Ab, 12.5,25 and 50ng/mL), and ZEN-Ab (concentration is followed successively by 50,100,200 and 400ng/mL) i.e. each four concentration, and (be followed successively by 250 with the concentration of biotinylation goat-anti mouse two anti-(Sec-Ab), 500,1000 and 2000ng/mL) solution does the chessboard titration, and it is mixed in (every kind of triplicate) in the PCR pipe, and do blank and negative control (each triplicate), guaranteeing to mix the back cumulative volume is 50-70 μ L.Each the 0.5-0.7 μ L of conjugate (guarantee in each reaction tube every kind microballoon number＞2000) that adds four kinds of comlete antigens after the high speed vortex vibration and microballoon then successively, and be provided with blank and negative control, in 37 ℃ of concussion 20-40min, carry out immune association reaction with aluminium-foil paper parcel back.
(2) after the vibration, to abandon supernatant behind 14000 * g high speed centrifugation 3-6min, add a certain proportion of SA-PE solution 50-70 μ L again, with the aluminium-foil paper parcel, in 37 ℃ of concussion 20-40min.
(3) open the suspending chip instrument according to normal procedure, preheating 30min, and select as required whether to proofread and correct.
(4) mixed solution after will vibrating is transferred in 96 orifice plates by the volume of 40-60 μ L.Put 96 orifice plates the detection platform of Luminex 100 systems into, set the reaction detection program, obtain meta fluorescent value (Median fluorescent intensitity abbreviates MFI as) after the detection by software kit.
(5) by the MFI value after extracting is handled and analyzed, draw the best effort concentration of four kinds of mycotoxin monoclonal antibodies and Sec-Ab.
The experiment in stage draws thus: the best effort concentration (correlated results is seen accompanying drawing 2) of four kinds of mycotoxin monoclonal antibodies and Sec-Ab is respectively: AFT-Ab 100ng/mL, DON-Ab 10ng/mL, T-2-Ab 25ng/mL, ZEN-Ab 200ng/mL, Sec-Ab 2000ng/mL.The result who comprehensively draws analyzes (correlated results is seen accompanying drawing 3) to the specificity of four kinds of mycotoxin antigen-antibodies, and the result shows that specificity is good between each antigen-antibody, and cross reaction does not take place.The hyperchannel typical curve had been done sufficient preparation when these results detected four kinds of mycotoxins simultaneously for going on foot drafting down.
The foundation of hyperchannel typical curve when embodiment 3 detects four kinds of mycotoxins simultaneously
Utilize coupling that the microballoon of comlete antigen is arranged, and the anti-best effort concentration of the high four kinds of mycotoxin monoclonal antibodies of the specificity to be detected after determining and biotinylation two, carry out the experiment in this stage.
Concrete steps are as follows:
(1) the first best effort concentration solution of four kinds of mycotoxin monoclonal antibodies of preparation, i.e. AFT-Ab 100ng/mL, DON-Ab 10ng/mL, T-2-Ab 25ng/mL, ZEN-Ab 200ng/mL, and it is mixed in the PCR pipe, guaranteeing to mix the back cumulative volume is 50-70 μ L.Then, each the 0.5-0.7 μ L of conjugate (guarantee in each reacting hole every kind microballoon number＞2000) that adds four kinds of comlete antigens after the high speed vortex vibration and microballoon successively, and be provided with blank, negative control and positive control, in 37 ℃ of concussion incubation 5-15min, carry out immune association reaction with aluminium-foil paper parcel back.
(2) after the vibration, add a series of mixed liquors that contain four kinds of targets, wherein the AFB1 concentration range is 0.0098-640ng/mL, DON is 0.1221-8000ng/mL, T-2 is 0.0002-10ng/mL, and ZEN is 0.0098-640ng/mL, guarantees that the cumulative volume of mixed liquor is 80-100 μ L.Behind the aluminium-foil paper parcel, in 37 ℃ of vibration 15-25min, reaction is at war with.
(3) after the vibration, 14000 * g high speed centrifugation 3-6min abandons supernatant, adds the optium concentration solution (being 2000ng/mL) of 50-70 μ L Sec-Ab again, in 37 ℃ of concussion incubation 15-25min, carries out immune response with aluminium-foil paper parcel back.
(4) after the vibration, abandon supernatant with the high speed centrifugation 3-6min of 14000 * g, add a certain proportion of SA-PE solution 50-70 μ L again, with aluminium-foil paper parcel back in 37 ℃ of vibration incubation 20-40min.
(5) open the suspending chip instrument according to normal procedure, preheating 30min, and select as required whether to proofread and correct.
(6) mixed solution after will vibrating is transferred in 96 orifice plates by the cumulative volume of 40-60 μ L.Put 96 orifice plates into detection platform, set the reaction detection program, obtain MFI after the detection by software kit.
(7) by the MFI value after extracting is handled and analyzed, draw out the hyperchannel typical curve of four kinds of mycotoxins of detection when being matrix with the antibody diluent.
The experimental result in stage is drawn out in antibody diluent thus, and the hyperchannel typical curve when detecting four kinds of mycotoxins is simultaneously seen accompanying drawing 4-1, and dependent equation and parameter thereof see Table 1.Setting up the suspension chip method that detects four kinds of mycotoxins in corn, the peanut actual sample simultaneously during these results are down and go on foot prepares.Embodiment 4 detects the foundation of four kinds of mycotoxin suspension chip methods in corn, the peanut simultaneously
Utilize the microballoon of coupling comlete antigen, the best effort concentration that definite four kinds of mycotoxin monoclonal antibodies and biotinylation two are anti-, and the hyperchannel typical curve that detects four kinds of mycotoxins when drawing out carries out the experiment in this stage.
Mainly consider that from drafting and the matrix effect of actual sample examination criteria curve concrete steps are as follows:
(1) corn and the peanut (local supermarket) buied are smashed with soy bean milk making machine, taken by weighing corn or the peanut powder of 1-3g, fully add 20% (corn) of 5-15mL or the methanol aqueous solution of 40% (peanut) behind the mixing.High speed vortex vibration 3-7min, fully mixing.
(2) with the solution behind the mixing under room temperature, the centrifugal 5-15min of 3000-5000 * g.
(3) get supernatant solution after centrifugal, carry out a certain proportion of dilution with antibody diluent, as negative treating fluid, can be described as the negative sample liquid of corn and the negative sample liquid of peanut.And draw multichannel typical curve with the negative sample liquid of corn and the negative sample liquid of peanut as dilution respectively, by to MFI
0(positive control) and IC
50Comparative analysis, judge whether that with this available suspending chip detects four kinds of mycotoxins in corn and the peanut.
After a series of optimization experiment, judge that corn and peanut negative sample liquid separately are to MFI
0And IC
50Influence (table 2) after as can be known: pre-treating method is to different mycotoxin MFI
0Certain influence is arranged, but to IC
50The value influence is not obvious, that is to say the sensitivity influence insensitive.So the negative sample liquid of corn and the negative sample liquid of peanut can replace antibody diluent conduct dilution separately, thereby the hyperchannel competition test curve (seeing accompanying drawing 4-2 and 4-3) with the drafting of suspending chip method, as seen from the figure: the competition trend of these curves be that the curve competition trend difference drawn of matrix is little with the antibody diluent, the correlation parameter of competition curve sees Table 1.Generally speaking, the sensitivity that the suspending chip method detects four kinds of mycotoxins can reach pg～ng level level, is 3～4 orders of magnitude between detection zone.
Embodiment 5 suspending chip methods detect accuracy and the repetitive research of corn, peanut
Mainly from the mark-on of actual sample being reclaimed accuracy and the repeatability of experiment decision method, concrete steps are as follows:
(1) take by weighing corn or the peanut powder of 1-3g, add four kinds of mycotoxin standard items simultaneously by certain concentration, vibration makes its abundant mixing at a high speed, adds 20% or 40% the methanol aqueous solution of 5-15mL then.High speed vortex vibration 3-7min makes its abundant mixing.
(2) with the solution behind the mixing under room temperature, with the centrifugal 5-15min of 3000-5000 * g.
(3) get supernatant solution after centrifugal, with antibody diluent it is carried out a certain proportion of dilution, as mark-on solution.Carry out mark-on according to embodiment 3 and detect, by calculate repeatability and the accuracy of evaluation (the present invention is directed to corn and peanut) in actual sample suspending chip method at the hyperchannel typical curve of drawing with negative corn-like liquid and negative peanut sample liquid.
The mark-on testing result of actual sample sees Table 3, and the recovery of standard addition of actual sample is all in the 80-120% scope as can be known, and the coefficient of variation all＜15% shows the suspending chip method accuracy height that the present invention sets up, good reproducibility.Draw thus: this method can quantitatively detect the content of four kinds of mycotoxins in corn and the peanut easy, quickly and efficiently simultaneously.
Table 3 suspending chip method detects the recovery of standard addition in corn and the peanut
Claims (12)
1. the suspension chip method that quantitatively detects of mycotoxin more than a kind is by carboxylated microballoon, be coupled to Protein Detection index on the microballoon and corresponding monoclonal antibody index thereof etc. and form.It is characterized in that the Protein Detection index of described microsphere surface comprises: the comlete antigen of total aflatoxin content, vomitoxin, T-2 toxin, four kinds of mycotoxins of zearalenone, all by EDC method covalent coupling in carboxylated microsphere surface.
2. claims 1 described carboxylated microballoon is that a kind of diameter is the polystyrene microsphere of 5.6 μ m, and microballoon has two kinds of fluorescent materials, according to the concentration proportioning difference of two kinds of fluorescent materials, microballoon can be divided into 100 kinds.Carboxyl on the microsphere surface carries out covalent bond by the amino on EDC method and the Protein Detection index, and is liquid-phase reaction system, is conducive to keep the biologically active of protein molecular.
3. the comlete antigen of four kinds of mycotoxins according to claim 1, namely the conjugate of four kinds of little molecules and bovine serum albumin(BSA) (BSA) is followed successively by: total aflatoxin content comlete antigen (AFT-BSA), vomitoxin comlete antigen (DON-BSA), T-2 toxin comlete antigen (T-2-BSA) and zearalenone comlete antigen (ZEN-BSA).
4. little molecule mouse resource monoclonal antibody according to claim 1 is followed successively by: aspergillus flavus resisting toxin total amount monoclonal antibody (AFT-Ab), emesis toxin monoclone antibody (DON-Ab), anti-T-2 toxin monoclone antibody (T-2-Ab), anti-zearalenone monoclonal antibody (ZEN-Ab).
5. four kinds of mycotoxin haptens according to claim 3 are aflatoxin B1 (AFB1), vomitoxin (DON), T-2 toxin (T-2), zearalenone (ZEN) successively.
6. each comlete antigen and microballoon coupling may further comprise the steps in the described suspending chip method of preparation claim 1:
(1) the carboxylated microballoon with blank takes out from 4 ℃ of refrigerators, in dark situation it is returned to room temperature, it is activated again;
(2) prepare each comlete antigen solution by the concentration of experimental design, by EDC method and carboxylated microballoon coupling, i.e. the conjugate of each comlete antigen and microballoon, and it is sealed;
(3) with after the vibration of conjugate vortex, with the suspending chip instrument it is carried out reading.Remaining wraps up with aluminium-foil paper, in 4 ℃ of storages.
Annotate: all operations process of the present invention is all carried out under dark situation.
7. according to claim 5, when carrying out the suspending chip experiment, should earlier conjugate and the used various damping fluids of suspending chip be taken out from 4 ℃ of refrigerators, enable after returning to room temperature.
8. of the present invention is the indirect competition immune response, mainly is divided into the work in five stages:
(1) four kind of mycotoxin comlete antigen respectively with the coupling of different coding microballoon;
Determining of (2) four kinds of mycotoxin monoclonal antibodies and biotinylation two anti-best effort concentration, and specific checking between the antigen-antibody;
The foundation of hyperchannel typical curve when (3) detecting four kinds of mycotoxins simultaneously;
(4) detect the foundation of four kinds of mycotoxin suspension chip methods in corn, the peanut simultaneously;
(5) the suspending chip method detects accuracy and the repetitive research of corn, peanut.
According to Claim 8 in (2), prepare each monoclonal antibody and the working concentration solution that biotinylation two resists by the concentration of setting, carry out chessboard optimization, draw the working concentration of each antibody the best, and each antigen-antibody carried out specific detection.
According to Claim 8 in (3), working concentration solution according to each the antibody the best after optimizing is prepared, and prepare each haptenic mixed solution by a series of concentration of setting, make itself and comlete antigen compete limited antibody combining site, draw out the hyperchannel typical curve that detects four kinds of mycotoxins simultaneously.
11. according to Claim 8 (4), corn and the peanut sample that will buy from the local market carry out pre-treatment with a certain proportion of methanol/water solution, and negative extract is diluted.Be dilution with the negative sample liquid of this corn and the negative sample liquid of peanut, by best effort concentration preparation antibody-solutions, and the haptens mixed liquor of a series of concentration, make the immune response that is at war with of itself and corresponding conjugate, drawing out in corn and the peanut hyperchannel typical curve that detects four kinds of mycotoxins, by to MFI
0(positive control) and IC
50Compare analysis, thereby matrix effect is estimated.
12. according to Claim 8 (5), analyze by the mark-on testing result to actual sample, the repeatability and the accuracy that estimate to detect can detect four kinds of mycotoxins in corn and the peanut sample simultaneously thereby draw the suspending chip method.
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| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
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| CN109164254A (en) * | 2018-09-21 | 2019-01-08 | 天津大学 | The platform building and preparation method of granules of hydrogel fines for mycotoxin detection |
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| CN112098661A (en) * | 2020-09-22 | 2020-12-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for simultaneously detecting five endocrine disruptors and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
| CN106932370B (en) * | 2017-03-07 | 2019-08-13 | 中国农业科学院油料作物研究所 | The synchronous chromatography time-resolved fluorescence kit for detecting five kinds of mycotoxins and application |
| CN109115739A (en) * | 2018-08-06 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of hole internal calibration test method |
| CN109115739B (en) * | 2018-08-06 | 2023-11-10 | 宙斯生命科技(常州)有限公司 | Method for testing in-hole calibration |
| CN109164254A (en) * | 2018-09-21 | 2019-01-08 | 天津大学 | The platform building and preparation method of granules of hydrogel fines for mycotoxin detection |
| CN110066403A (en) * | 2019-03-10 | 2019-07-30 | 天津大学 | The preparation method of Fungi Mycotoxin identification photon crystal water gel |
| CN112098661A (en) * | 2020-09-22 | 2020-12-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method and kit for simultaneously detecting five endocrine disruptors and application |
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