CN102798714A - Immuno-chip for detecting multiple fungal toxins and preparation method thereof - Google Patents
Immuno-chip for detecting multiple fungal toxins and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method for an immuno-chip capable of detecting multiple fungal toxins (aflatoxin B1, aflatoxin M1, vomitoxin, ochratoxin A, T-2 toxin and zearalenone) simultaneously and a detection technique of the immuno-chip. Complete antigen sites of the toxins are prepared on the chip, enter an agarose polymer layer of an activated aldehyde group in a dispersing mode, and are covalently coupled to the surface of the chip; and the technical research of the immuno-chip is carried out by a competition method, namely during detection, monoclonal antibodies of the toxins and a standard substance are mixed and added to the chip, corresponding to-be-detected substance antigens and micromolecules compete the monoclonal antibodies, the second antibody of a Cy3 label is added, a to-be-detected substance antigen-monoclonal antibody-second antibody compound is formed, and fluorescent signals are weakened along the increase of the concentration of the micromolecules, so that the detection aim is fulfilled. By the technique, the sensitivity reaches level pg-ng, results are stable and reliable, the consumption of samples is low, the detection is simple and quick, and a new method for detecting multiple toxins is provided.
Description
Technical field
The present invention relates to a kind of preparation method of immuno-chip, particularly related to a kind of immuno-chip detection technique that detects multiple mycotoxin.
Background technology
Mycotoxin (Mycotoxin) is a kind of in the biotoxin, is the secondary metabolite that thread micromycete (these fungies constitute a serious threat to the disease of animal and human's class) produces.At present, known have a different mycotoxin of kind more than 350.Usually; Mycotoxin pollutes cereal crops and animal feed; And then pollute animal foods such as meat, egg, milk; Get into human biologic chain, not only can cause the mould reduction that loses rotten, loss of nutritional ingredients and product quality of agricultural product, also can be through the synthetic inhibition of DNA, RNA, protein and various enzymes in the humans and animals body and pair cell structural damage being caused teratogenesis, mutagenesis and serious harm such as carcinogenic.Therefore, in global environment and food-safety problem, receive much concern.
Along with international and domestic reinforcement day by day to the food security attention degree, and in view of the harm that mycotoxin brought, in agricultural product and food security index, the content of mycotoxin has become important detection index.That research both at home and abroad mainly concentrates on is common in agricultural product and the feed, on tens kinds of bigger mycotoxins of mankind's harm, they generally have strong toxicity simultaneously and pollute characteristics such as frequency height.According to this, the target that the present invention studied is six kinds of mycotoxins, comprise AFB1 (Aflatoxin B1, AFB1), it is acknowledged as carcinogenicity and the strongest toxin of toxicity, target organ is a liver, can suppress growth of animal, teratogenesis etc.; Aflatoxin M 1 (Aflatoxin M1, AFM1), that its toxicity mainly shows as is carcinogenic, mutagenicity etc.; Vomitoxin, (Deoxynivalenol DON), has very strong cytotoxicity, and synthesizing of CKIs matter also can cause Reproductive and developmental toxicity, immunotoxicity, carcinogenicity etc. to have another name called deoxynivalenol; Ochratoxin A (Ochratoxin A, OTA), its target organ is a kidney, shows the damage of tubular degeneration and function, carcinogenic teratogenesis etc.; The T-2 toxin (T-2 toxin, T-2), but its CKIs matter, DNA's is synthetic, strong immunotoxicity, carcinogenic teratogenesis has lethal effect and to the toxicity of Skin Cell and heredity; Zearalenone, (Zearalenone ZEN) mainly acts on reproductive system, can cause that people and animals are taken place by the hyperfunction disease of estrogen, has than Johnson & Johnson and grows toxicity and teratogenesis etc. to have another name called the F-2 toxin.
At present, detecting the most frequently used method of mycotoxin both at home and abroad is high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA), and the former detection sensitivity is high, but sample pre-treatments is loaded down with trivial details, complicated operation, time are long, and equipment needed thereby is costliness; The latter is easy and simple to handle, quick, but can only accomplish a kind of detection of mycotoxin at every turn, and during for the check fee of multiple toxin, effort, workload is huge.
Immuno-chip (Immunochip) is a kind of in the protein-chip; Antigen or antibody microarray by being fixed on the different supporting dielectrics are formed; The position of fixed member and composition are known in the array, react with the probe on corresponding antibody or antigen and the chip, and two anti-the carrying out that will be marked with fluorescent material, enzyme or chemiluminescent substance etc. again react the second time; Detect through specific scanister then; Extract the scanning result data, and it is carried out Treatment Analysis, thereby reach the purpose that determinand is detected with Origin 8.0 softwares.
The immuno-chip that the multiple mycotoxin of preparation detects has advantages such as high flux, easy and simple to handle, good reproducibility, sensitivity height; Be applicable to the fast quantification of mycotoxin micromolecule chemical pollutant 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 defective, a kind of immuno-chip detection technique of six kinds of mycotoxins fast simple to operate, sensitive is provided to existing detection method existence.
Another object of the present invention has provided the preparation method of immuno-chip when detecting six kinds of mycotoxins simultaneously.
Technical scheme of the present invention is summarized as follows:
(1) confirms the best effort concentration of six kinds of mycotoxin antigens.
(2) confirm the best effort concentration of six kinds of mycotoxin antibody and the detection of antigen and antibody specific.
(3) detect the drafting of six kinds of mycotoxin typical curves simultaneously.
(4) mensuration of multiple mycotoxin in the testing sample.
Preferably step (1) confirms that the best effort concentration of six kinds of mycotoxin antigens is: the working concentration of six kinds of mycotoxin comlete antigens respectively is set at six kinds of different concentration gradients; And be formed on the chip with time point with three kinds of contrasts; Prepare the immuno-chip in this stage, identify through scanner; Again with the binding immunoassay reaction that is at war with of the mouse resource monoclonal antibody of described six kinds of mycotoxins; Obtain the best effort concentration and the more excellent working concentration of antibody of six kinds of mycotoxin antigens, prepare for going on foot the best effort concentration of confirming six kinds of mycotoxin antibody down.
Said step (2) confirms that the best effort concentration of six kinds of mycotoxin antibody and the detection of antigen and antibody specific are: the best effort concentration solution of preparing six kinds of mycotoxin antigens earlier; Be formed on the chip with time point with three kinds of contrast liquid; Prepare the immuno-chip in this stage, identify through scanner; Again that described six kinds of mycotoxin antibody are more excellent working concentration solution (each four concentration gradient) carries out immune response, obtains the best effort concentration of six kinds of mycotoxin antibody, and the drafting of typical curve is prepared when detecting six kinds of mycotoxins simultaneously for the following step.And the result that integrating step (1) draws analyzes the specificity of six kinds of mycotoxin antigen-antibodies.
Said step (3) detects being plotted as of six kinds of mycotoxin typical curves simultaneously: the best effort concentration solution of preparing six kinds of mycotoxin antigens earlier; Be formed on the chip with time point with three kinds of contrast liquid; Prepare the immuno-chip in this stage, identify through scanner; The binding immunoassay reaction that is at war with of again that described six kinds of mycotoxin antibody are best working concentration solution obtains on a chip, detecting simultaneously the typical curve of six kinds of mycotoxins, prepares for going on foot in the testing sample mensuration of multiple mycotoxin down.
Being determined as of multiple mycotoxin in said step (4) testing sample: the best effort concentration solution of six kinds of mycotoxin antigens of preparation earlier, be formed on the chip with time point with three kinds of contrast liquid, prepare the immuno-chip in this stage, identify through scanner; The binding immunoassay reaction that is at war with of again that described six kinds of mycotoxin antibody are best working concentration solution, obtain on a chip to tap water in six kinds of mycotoxins carry out mark-on and detect.
Through a series of optimization experiment, the present invention can detect the mark-on content of six kinds of mycotoxins in the water easy, effectively, and sensitivity can reach pg～ng level, is 2～3 one magnitude between detection zone, and it is less that result and actual value are compared deviation.
Description of drawings
Fig. 1 is the design of the layout and the inferior array of every chip.
Wherein, A figure is the topological chip plan after agarose is modified, and every chip contains 12 independently inferior arrays; B figure is the design drawing of phase one work each probe when promptly confirming six kinds of mycotoxin antigen optium concentrations, and probe mainly comprises: six kinds of mycotoxin comlete antigens (AFT-BSA, AFM1-BSA; DON-BSA; OTA-OVA, T-2-BSA, ZEN-BSA) working solution and three kinds of contrast working fluid (Cy3-BSA; Blank, mouse-IgG); The design drawing of each probe when C figure is back three phases work; This moment, probe was optium concentration working fluid and three kinds of contrast working fluids of six kinds of mycotoxin comlete antigens, and the best effort concentration of each toxin antigen is followed successively by: AFT-BSA 0.2mg/mL, AFM1-BSA 0.1mg/mL, DON-BSA 2.0mg/mL, OTA-OVA 2.0mg/mL, T-2-BSA 0.2mg/mL, ZEN-BSA 1.0mg/mL.
Fig. 2 is the operating process of immuno-chip detection technique.
Fig. 3 is for optimizing the optium concentration result of six kinds of mycotoxin antigens.
Wherein, 3-1A (AFT), 3-2A (AFM1), 3-3A (DON), 3-4A (OTA), 3-5A (T-2), 3-6A (ZEN) are respectively scintigram behind antibody working fluid and each probe hybridization of six kinds of variable concentrations of six kinds of mycotoxins; 3-1B (AFT), 3-2B (AFM1), 3-5B (T-2) figure are respectively the antibody of six kinds of variable concentrations and corresponding antigen hybrid curve figure.
Fig. 4 is for optimizing the optium concentration result of six kinds of mycotoxin antibody.
Wherein, 4-1A (AFT), 4-2A (AFM1) 4-3A (DON), 4-4A (OTA), 4-5A (T-2), 4-6A (ZEN) are each mycotoxin antibody working fluid hybridization four kinds of variable concentrations and more excellent back scintigrams.Accordingly, 4-1B (AFT), 4-2B (AFM1), 4-3B (DON), 4-4B (OTA), 4-5B (T-2), 4-6B (ZEN) are each antigen-antibody hybrid curve figure.
Fig. 5 is the specific detection result of six kinds of mycotoxin antigen-antibodies.
Fig. 6 detects the drafting of six kinds of mycotoxin typical curves simultaneously for the immuno-chip method.
Wherein, A figure be the immuno-chip method when detecting six kinds of mycotoxins simultaneously the mycotoxin micromolecule compete immune response scintigram as a result with comlete antigen; B figure is six kinds of typical curves that utilization Origin 8.0 softwares are drawn out.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is done further elaboration.Below embodiment be for the ease of understanding the present invention better, but do not limit the present invention.
Embodiment 1:
Experiment material is following:
AFB1 (Aflatoxin B1; AFB1), vomitoxin (Deoxynivalenol; DON), ochratoxin A (Ochratoxin A or Benaene Free; OTA), the T-2 toxin (T-2 Toxin, T-2), zearalenone (Zearalenone, ZEN): Fermentek Ltd.; Aflatoxin M 1 (Aflatoxin M, AFM1): Supelco company; Aflatoxin M 1 comlete antigen (AFM1-BSA): Sigma company; Total aflatoxin content comlete antigen (AFT-BSA); Vomitoxin comlete antigen (DON-BSA); Ochratoxin A comlete antigen (OTA-OVA); T-2 toxin comlete antigen (T-2-BSA); Zearalenone comlete antigen (ZEN-BSA); Aspergillus flavus resisting toxin total amount monoclonal antibody (AFT-Ab); Aflatoxin M 1 antibody (AFM1-Ab); Emesis toxin monoclone antibody (DON-Ab); Anti-ochratoxin A monoclonal antibody (OTA-Ab); Anti-T-2 toxin monoclone antibody (T-2-Ab); Anti-zearalenone monoclonal antibody (ZEN-Ab): Huaan, Beijing Mai Ke Bioisystech Co., Ltd; Goat anti-mouse IgG (H+L), Cy3: Beijing health is the century bio tech ltd; Brilliant core
protein chip sampling liquid-A: Beijing Boao Biological Co., Ltd; Antibody diluent, 100% sheep blood serum, mouse-IgG, BSA-Cy3 all have Bo Ao company to provide.It is pure that other chemical reagent is homemade analysis, and used pure water is the Milli-Q ultrapure water.
Decolorization swinging table: its woods Bel instrument Manufacturing Co., Ltd of Haimen City; Brilliant core
Polymer three-dimensional substrate G (being the agarose chip), brilliant core
PersonalArrayer
TM16 people's point sample instruments, brilliant core
LuxScan
TM10K-A micro-array chip scanner, brilliant core
SlideWasher
TM8 chips are washed dried appearance: Beijing Boao Biological Co., Ltd.
(1) confirms the best effort concentration of six kinds of mycotoxin antigens
1) preparation of chip
Concentration 0.00625～the 0.2000mg/mL of six kinds of mycotoxin comlete antigens is prepared, and prepare three kinds of contrast liquid (Cy3-BSA, sampling liquid, 0.5mg/mL mouse-IgG).Then, it is changed in 384 orifice plates, low-temperature dark is preserved, and is subsequent use.
2. to PersonalArrayer
TMThe running program of 16 people's point sample instruments is provided with, and guarantees that its temperature is 24～26 ℃, and humidity is 22～37%.Through point sample instrument each probe points is formed on the chip after agarose is modified.
3. with the chip behind the point sample in the workshop of high-cleanness, high overnight treatment, make probe stationary on chip, for immune response is prepared.
4. the chip after will fixing scans, and it is identified, is undertaken by design proposal to guarantee that probe is arranged.Then, be positioned in the chip storage box, vacuumize the packing back and preserve, can preserve 6 months in 4 ℃ of refrigerators.When launching, should earlier it be returned to room temperature.
2) immune response
1. sealing
Take out the chip hybridization box, in hybridizing box, add 200 μ L distilled water or tap water, so that wet environment is provided for hybridization reaction.
Take out the chip of desire hybridization the chip storage box after returning to room temperature, chip front side upwards carefully is put on the carriage of hybridizing box.One of each inferior array jiao slowly adds 20～40 μ L confining liquids (i.e. 100% sheep blood serum) from chip, covers the reaction lid, in 37 ℃ of constant temperature ovens, hatches 30～60min.
2. the cleaning of chip is with dry
Chip after taking-up is hatched gets rid of excess liquid, and chip is put in the chip cleaning box that 1 ‰ PBST solution are housed, and the confining liquid that flush away is residual takes out chip, outwells the PBST solution of using; 1 ‰ clean PBST solution are poured in the cleaning box, put into chip, and it is positioned over to rock on the shaking table cleans 5～10min, take out chip in order to centrifugal drying.
Chip is put in Slide Washer
TM8 chips are washed in the dried appearance and are dried, and centrifugal condition is 1500rpm, 5min.During placement, should chip tag inwardly be put in the hydro-extractor, and note trim.
3. first step immune response
Chip behind the centrifuge dripping is positioned in the chip hybridization box, and six kinds of mycotoxins having prepared one anti-working fluid 0.3125～10.0000 μ g/mL is added by the inferior array of 20～40 μ L/, in 37 ℃ of constant temperature ovens, hatch 30～60min.
4. the cleaning of chip is with dry
The biconditional operation step 2..
5. the second step immune response
Prepare two anti-working fluids by a certain percentage, under dark situation, the inferior array of 20～40 μ L/ is added, put into the hybridization reaction box, cover lid, and in 37 ℃ of constant temperature ovens, hatch 30～60min.
6. the cleaning of chip is with dry
The biconditional operation step 2..
7. scanner uni interpretation of result
Scan with the chip of chip scanner after, extract data and it is handled and analyzes hybridization.During scanning, excitation wavelength is 532nm, and resolution is 10 μ m, and adjustable sweep parameter is photomultiplier PMT (450～950 is adjustable continuously), power P ower (1～100%).
In conjunction with ELISA testing result (seeing table 1); Each antigen optium concentration of deducibility is respectively: AFT-BSA 0.2mg/mL, AFM1-BSA 0.1mg/mL, DON-BSA 2.0mg/mL, OTA-OVA 2.0mg/mL, T-2-BSA 0.2mg/mL, ZEN-BSA 1.0mg/mL, the more excellent concentration of each antibody is followed successively by AFT-Ab0.04～5.00 μ g/mL, AFM1-Ab 0.04～5.00 μ g/mL, DON-Ab 0.08～10.00 μ g/mL, OTA-Ab 0.16～20.00 μ g/mL, T-2-Ab 0.08～10.00 μ g/mL, ZEN-Ab 0.16～20.00 μ g/mL.
(2) confirm the best effort concentration of six kinds of mycotoxin antibody and the detection of antigen and antibody specific
1) preparation of chip
1. the comlete antigen of six kinds of mycotoxins is prepared by optium concentration; Be AFT-BSA 0.2mg/mL, AFM1-BSA 0.1mg/mL, DON-BSA 2.0mg/mL, OTA-OVA 2.0mg/mL, T-2-BSA 0.2mg/mL, ZEN-BSA 1.0mg/mL; And prepare three kinds of blank liquid (Cy3-BSA
aThe ELISA testing result of six kinds of mycotoxins of table 1
A: this result is provided by biological company limited of Huaan, Beijing wheat section.
Sampling liquid, 0.5mg/mL mouse-IgG).Then, it is changed in 384 orifice plates, low-temperature dark is preserved, and is subsequent use.
2. the running program to point sample instrument is provided with, and guarantees that its temperature is 24～26 ℃, and humidity is 22～37%.Through point sample instrument each probe points is formed on the chip after agarose is modified.
3. with the chip behind the point sample in the workshop of high-cleanness, high overnight treatment, make probe stationary on chip, for immune competitive reaction is prepared.
4. the chip after will fixing scans, and it is identified, is undertaken by scheme of designing to guarantee that probe is arranged.Then, be positioned in the chip storage box, vacuumize the back and preserve, can preserve 6 months in 4 ℃ of refrigerators.When launching, should earlier it be returned to room temperature.
2) immune competitive reaction
1. sealing
Take out the chip hybridization box, in hybridizing box, add 200 μ L distilled water or tap water, so that wet environment is provided for hybridization reaction.
Take out the chip of desire hybridization the chip storage box after returning to room temperature, chip front side upwards carefully is put on the carriage of hybridizing box.One of each inferior array jiao slowly adds 20～40 μ L confining liquids (i.e. 100% sheep blood serum) from chip, covers the reaction lid, in 37 ℃ of constant temperature ovens, hatches 30～60min.
2. the cleaning of chip is with dry
Chip after taking-up is hatched gets rid of excess liquid, and chip is put in the chip cleaning box that 1 ‰ PBST solution are housed, and the confining liquid that flush away is residual takes out chip, outwells the PBST solution of using; 1 ‰ clean PBST solution are poured in the cleaning box, put into chip, and it is positioned over to rock on the shaking table cleans 5min, take out chip in order to centrifugal drying.
Chip is put in SlideWasher
TM8 chips are washed in the dried appearance and are dried, and centrifugal condition is 1500rpm, 5min.During placement, should chip tag inwardly be put in the hydro-extractor, and note trim.
3. first step immune response
Chip behind the centrifuge dripping is positioned in the chip hybridization box, and the working fluid of the more excellent concentration that six kinds of mycotoxins having prepared are anti-adds by the inferior array of 20～40 μ L/, in 37 ℃ of constant temperature ovens, hatch 30～60min.
4. the cleaning of chip is with dry
The biconditional operation step 2..
6. the second step immune response
Prepare two anti-working fluids by a certain percentage, under dark situation, the inferior array of 20～40 μ L/ is added, put into the hybridization reaction box, cover lid, and in 37 ℃ of constant temperature ovens, hatch 30～60min.
6. the cleaning of chip is with dry
The biconditional operation step 2..
7. scanner uni interpretation of result
Scan with the chip of chip scanner after, extract data and it is handled and analyzes hybridization.During scanning, excitation wavelength is 532nm, and resolution is 10 μ m, and adjustable sweep parameter is photomultiplier PMT (450～950 is adjustable continuously), power P ower (1～100%).
Can know that by interpretation of result the best effort concentration of six kinds of mycotoxin antibody is followed successively by: AFT-Ab 1.00 μ g/mL, AFM1-Ab 0.50 μ g/mL, DON-Ab 1.00 μ g/mL, OTA-Ab 0.10 μ g/mL, T-2-Ab1.00 μ g/mL, ZEN-Ab 1.00 μ g/mL.
Result in conjunction with (1) stage can know, the specificity between six kinds of mycotoxin antigen-antibodies is seen Figure of description 5.Hence one can see that: except that AFT-BSA and AFM1-Ab, AFM1-BSA and AFT-BSA have the cross reaction, other antigen and antibody specific is good.But AFT-BSA and AFM1-Ab cross reacting rate only have 57.10%, so the present invention adopts the AFT antigen-antibody to detect AFB1, the AFM1 antigen-antibody detects AFM1.
(3) detect the drafting of six kinds of mycotoxin typical curves simultaneously
1) preparation of chip
With 1 in (2) among the embodiment 1).
2) immune competitive reaction
1. sealing
Take out the chip hybridization box, in hybridizing box, add 200 μ L distilled water or tap water, so that wet environment is provided for hybridization reaction.
Take out the chip of desire hybridization the chip storage box after returning to room temperature, chip front side upwards carefully is put on the carriage of hybridizing box.One of each inferior array jiao slowly adds 20～40 μ L confining liquids (i.e. 100% sheep blood serum) from chip, covers the reaction lid, in 37 ℃ of constant temperature ovens, hatches 30～60min.
2. the cleaning of chip is with dry
Chip after taking-up is hatched gets rid of excess liquid, and chip is put in the chip cleaning box that 1 ‰ PBST solution are housed, and the confining liquid that flush away is residual takes out chip, outwells the PBST solution of using; 1 ‰ clean PBST solution are poured in the cleaning box, put into chip, and it is positioned over to rock on the shaking table cleans 5min, take out chip in order to centrifugal drying.
Chip is put in SlideWasher
TM8 chips are washed in the dried appearance and are dried, and centrifugal condition is 1500rpm, 5min.During placement, should chip tag inwardly be put in the hydro-extractor, and note trim.
3. first step immune response
Prepare by separately final concentration AFT-Ab 1.00 μ g/mL, AFM1-Ab 0.50 μ g/mL, DON-Ab 1.00 μ g/mL, OTA-Ab 0.10 μ g/mL, T-2-Ab 1.00 μ g/mL, ZEN-Ab 1.00 μ g/mL by an anti-hybrid working liquid of six kinds of mycotoxins.The standard items hybrid working liquid of six kinds of mycotoxins is prepared by the finite concentration gradient.Get 20～40 μ L, one anti-hybrid working liquid and standard items hybrid working liquid mixing, under 37 ℃ of constant temperature and humidity conditions, hatch 10～20min in advance.
Chip behind the centrifuge dripping is positioned in the chip hybridization box, again the hybrid working liquid after hatching is added by the inferior array of 20～40 μ L/, in 37 ℃ of constant temperature ovens, hatch 30～60min.
4. the cleaning of chip is with dry
The biconditional operation step 2..
5. the second step immune response
Prepare two anti-working fluids by a certain percentage, under dark situation, the inferior array of 20～40 μ L/ is added, put into the hybridization reaction box, cover lid, and in 37 ℃ of constant temperature ovens, hatch 30～60min.
6. the cleaning of chip is with dry
The biconditional operation step 2..
7. scanner uni interpretation of result
Scan with the chip of chip scanner after, extract data and it is handled and analyzes hybridization.During scanning, excitation wavelength is 532nm, and resolution is 10 μ m, and adjustable sweep parameter is photomultiplier PMT (450～950 is adjustable continuously), power P ower (1～100%).Its result is shown in table 2 and Figure of description 6.
Hence one can see that: remove AFB1, AFM1, ZEN competition test curve coefficient of determination R in the curvilinear equation
2Outside＜0.99, other linear fits are good.This method detection sensitivity can reach pg～ng level level, is 2～3 one magnitude between detection zone.
Competition result when table 2 immuno-chip method detects six kinds of mycotoxins simultaneously
(4) mark-on of six kinds of mycotoxins detects in the tap water
1) preparation of chip
With 1 in (2) among the embodiment 1).
2) immune competitive reaction
1. sealing
Take out the chip hybridization box, in hybridizing box, add 200 μ L distilled water or tap water, so that wet environment is provided for hybridization reaction.
Take out the chip of desire hybridization the chip storage box after returning to room temperature, chip front side upwards carefully is put on the carriage of hybridizing box.One of each inferior array jiao slowly adds 20～40 μ L confining liquids (i.e. 100% sheep blood serum) from chip, covers the reaction lid, in 37 ℃ of constant temperature ovens, hatches 30～60min.
2. the cleaning of chip is with dry
Chip after taking-up is hatched gets rid of excess liquid, and chip is put in the chip cleaning box that 1 ‰ PBST solution are housed, and the confining liquid that flush away is residual takes out chip, outwells the PBST solution of using; 1 ‰ clean PBST solution are poured in the cleaning box, put into chip, and it is positioned over to rock on the shaking table cleans 5min, take out chip in order to centrifugal drying.
Chip is put in SlideWasher
TM8 chips are washed in the dried appearance and are dried, and centrifugal condition is 1500rpm, 5min.During placement, should chip tag inwardly be put in the hydro-extractor, and note trim.
3. compete the binding immunoassay reaction
Prepare by separately final concentration AFT-Ab 1.00 μ g/mL, AFM1-Ab 0.50 μ g/mL, DON-Ab 1.00 μ g/mL, OTA-Ab 0.10 μ g/mL, T-2-Ab 1.00 μ g/mL, ZEN-Ab 1.00 μ g/mL by an anti-hybrid working liquid of six kinds of mycotoxins.The standard items hybrid working liquid of six kinds of mycotoxins is pressed sample 1 (blank sample; Be tap water), sample 2 (containing AFB1 1.5ng/mL, AFM1 1.5ng/mL, DON 60ng/mL, OTA 120ng/mL, T-23ng/mL, ZEN 3ng/mL), sample 3 (containing AFB13ng/mL, AFM1 3ng/mL, DON 120ng/mL, OTA 240ng/mL, T-2 6ng/mL, ZEN12ng/mL) prepare, this moment, dilution was a tap water.Get anti-hybrid working liquid and standard items hybrid working liquid (cumulative volume 40～60 μ L) mixing, under 37 ℃ of constant temperature and humidity conditions, hatch 10～20min in advance.
Chip behind the centrifuge dripping is positioned in the chip hybridization box, again the hybrid working liquid after hatching is added by the inferior array of 20～40 μ L/, in 37 ℃ of constant temperature ovens, hatch 30～60min.
4. the cleaning of chip is with dry
The biconditional operation step 2..
5. immunoreactive beacon
Prepare two anti-working fluids by a certain percentage, under dark situation, the inferior array of 20～40 μ L/ is added, put into the hybridization reaction box, cover lid, and in 37 ℃ of constant temperature ovens, hatch 30～60min.
6. the cleaning of chip is with dry
The biconditional operation step 2..
8. scanner uni interpretation of result
Scan with the chip of chip scanner after, extract data and it is handled and analyzes hybridization.During scanning, excitation wavelength is 532nm, and resolution is 10 μ m, and adjustable sweep parameter is photomultiplier PMT (450～950 is adjustable continuously), power P ower (1～100%).
By interpretation of result: the mark-on testing result of six kinds of mycotoxins is seen table 3 in the tap water.
The mark-on testing result summary sheet of six kinds of mycotoxins in table 3 tap water
-: do not detect.
Can be known that by table 3 recovery of standard addition of DON is respectively 135.9% and 60.9% in sample 2 and 3, the recovery of standard addition of OTA is 60.4% in the sample 3, the recovery of standard addition of various targets is all 80～120% in other samples.This shows that the recovery of standard addition of six kinds of mycotoxins is better in the immuno-chip while test sample, can tentatively be used for the detection to actual sample.
Claims (9)
1. the immuno-chip of mycotoxin detection by quantitative more than a kind is made up of Protein Detection index and various contrast index that 3D activation aldehyde radical agarose chip and array point are formed on the chip.It is characterized in that; Protein Detection index on the described chip surface comprises: total aflatoxin content, aflatoxin M 1, vomitoxin, ochratoxin A, T-2 toxin, zearalenone the comlete antigen of totally six kinds of mycotoxins, through the activation covalent coupling in chip surface.
2. claims 1 described agarose chip is on surface of glass slide, to have modified one deck 3D structural polymer that contains the activation aldehyde radical as thin as a wafer, i.e. Ago-Gel.Behind the point sample, comlete antigen albumen probe sample gets into the Ago-Gel polymeric layer through the mode of diffusion, and encapsulates on slide with activation aldehyde radical covalent coupling.Because its surperficial polymkeric substance is that the protein molecular that is fixed in substrate surface provides hydrophilic environment, helps keeping the activity of protein molecular.
3. the comlete antigen of six kinds of mycotoxins according to claim 1; Promptly the conjugate of six kinds of micromolecule and bovine serum albumin(BSA) (BSA) or oralbumin (OVA) is followed successively by: total aflatoxin content comlete antigen (AFT-BSA), aflatoxin M 1 comlete antigen (AFM1-BSA), vomitoxin comlete antigen (DON-BSA), ochratoxin A comlete antigen (OTA-OVA), T-2 toxin comlete antigen (T-2-BSA), zearalenone comlete antigen (ZEN-BSA).
Described three kinds of contrast liquid are followed successively by: BSA-Cy3 working fluid (the point sample contrast has positioning action concurrently), sampling liquid (blank), 0.5mg/mL mouse-IgG working fluid (hybridization contrast).
4. micromolecule mouse resource monoclonal antibody according to claim 1 is followed successively by: aspergillus flavus resisting toxin total amount monoclonal antibody (AFT-Ab), aspergillus flavus resisting toxin M1 monoclonal antibody (AFM1-Ab), emesis toxin monoclone antibody (DON-Ab), anti-ochratoxin A monoclonal antibody (OTA-Ab), anti-T-2 toxin monoclone antibody (T-2-Ab), anti-zearalenone monoclonal antibody (ZEN-Ab).
5. six kinds of mycotoxin haptens according to claim 3 are AFB1 (AFB1), aflatoxin M 1 (AFM1), vomitoxin (DON), ochratoxin A (OTA), T-2 toxin (T-2), zearalenone (ZEN) successively.
6. the method for preparing the described immuno-chip of claim 1 may further comprise the steps:
(1) prepares the coating buffer of each toxin comlete antigen by the concentration of experimental design, and prepare the working fluids of three kinds of contrast liquid (being point sample contrast, blank, hybridization contrast), promptly prepare the probe coating buffer;
(2) the probe coating buffer is transferred in 384 orifice plates, the specific program through point sample instrument carries out a system to it;
(3) chip after the special workplace of high-cleanness, high is to point sample carries out vacuum drying, so that it is fixed.After chip after fixing vacuumizes packing, can 4 ℃ deposit 6 months.
7. according to claim 5, when immuno-chip uses, should earlier chip storage box be taken out from 4 ℃ of refrigerators, launch again after making it restore to room temperature.
8. what the present invention adopted is the competitive immunoassay method, mainly is divided into the work of four-stage:
(1) the best of confirming six kinds of mycotoxin antigens encapsulates concentration.
(2) confirm the best effort concentration of six kinds of mycotoxin antibody and the specific detection of antigen-antibody.
(3) detect the drafting of six kinds of mycotoxin typical curves simultaneously.
(4) mensuration of multiple mycotoxin in the testing sample.
9. immuno-chip preparation method who detects multiple mycotoxin simultaneously, its characteristic comprises:
(1) competition binding immunoassay reaction
The reaction of competition binding immunoassay is meant that each the anti-hybrid working liquid (the inferior array of 20～40 μ L/) with standard items with multiple mycotoxin is added on the chip simultaneously, makes the comlete antigen of multiple mycotoxin combine corresponding monoclonal antibody with the competition of micromolecule standard items.
When the best of confirming multiple mycotoxin antigen-antibody encapsulated with working concentration, institute added liquid and is each working fluid of resisting; When the mark-on of each toxin in the typical curve of drawing multiple mycotoxin and the tap water detects, an anti-mixed liquor with standard items of multiple mycotoxin is hatched 10～20min earlier, add on the chip then, and hatch 30～60min in 37 ℃ of constant temperature and humidities.Clean and dry chip, subsequent use.
(2) immunoreactive beacon
Beacon is meant that on chip, adding two of Cy3 mark resists.(V: ratio V) was prepared two anti-working fluids (narrating unclear), adds by the inferior array of 20～40 μ L/, and hatches 20～40min in 37 ℃ of constant temperature and humidities in 1: 400.Clean and dry chip, subsequent use.
(3) scanning chip and interpretation of result
Use LuxScan
TMThe chip of 10K-A micro-array chip scanner after to hybridization scans, and extracts data and it is handled and analyzes.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014190944A1 (en) * | 2013-05-31 | 2014-12-04 | 南京祥中生物科技有限公司 | Biochip and method for simultaneously and visually detecting multiple antibiotics, illegal additives, and biotoxins |
| CN104569381A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method and enzyme linked immunosorbent assay kit for detecting aflatoxin M1 |
| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN107991478A (en) * | 2016-10-27 | 2018-05-04 | 广东交通职业技术学院 | A kind of surface plasmon resonance biosensor and its method applied to ZEN immune detections |
| CN108226102A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Detect the time-resolved fluoroimmunoassay kit of Aflatoxins M1 |
| WO2021093886A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence kit for synchronously detecting diacetoxyscirpenol, deoxynivalenol, and t-2 toxin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1627072A (en) * | 2003-12-11 | 2005-06-15 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Protein chip, preparation method and usage |
| RU2361203C1 (en) * | 2007-10-08 | 2009-07-10 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Method of determining quality of milk and dairy products |
-
2011
- 2011-05-24 CN CN2011101339635A patent/CN102798714A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1627072A (en) * | 2003-12-11 | 2005-06-15 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Protein chip, preparation method and usage |
| RU2361203C1 (en) * | 2007-10-08 | 2009-07-10 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Method of determining quality of milk and dairy products |
Non-Patent Citations (2)
| Title |
|---|
| 万亮: "三种真菌毒素蛋白质微阵列检测方法的初步研究以及抗四环素多克隆抗体的制备", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 姬华，等: "免疫学技术检测真菌毒素的研究进展", 《粮油加工》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014190944A1 (en) * | 2013-05-31 | 2014-12-04 | 南京祥中生物科技有限公司 | Biochip and method for simultaneously and visually detecting multiple antibiotics, illegal additives, and biotoxins |
| CN104569381A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method and enzyme linked immunosorbent assay kit for detecting aflatoxin M1 |
| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN107991478A (en) * | 2016-10-27 | 2018-05-04 | 广东交通职业技术学院 | A kind of surface plasmon resonance biosensor and its method applied to ZEN immune detections |
| CN108226102A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Detect the time-resolved fluoroimmunoassay kit of Aflatoxins M1 |
| WO2021093886A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence kit for synchronously detecting diacetoxyscirpenol, deoxynivalenol, and t-2 toxin |
| US11815511B2 (en) | 2019-11-15 | 2023-11-14 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, deoxynivalenol, and T-2 toxin |
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