CN109765375A - Three mycotoxin colloidal gold immune chromatography tests and preparation method - Google Patents
Three mycotoxin colloidal gold immune chromatography tests and preparation method Download PDFInfo
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- CN109765375A CN109765375A CN201910023101.3A CN201910023101A CN109765375A CN 109765375 A CN109765375 A CN 109765375A CN 201910023101 A CN201910023101 A CN 201910023101A CN 109765375 A CN109765375 A CN 109765375A
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Abstract
The invention discloses a kind of three mycotoxin colloidal gold immune chromatography tests, including according to the sequentially connected sample pad in sample flow direction, label pad, basement membrane and water absorption pad；The label pad includes the first gold pad and the second gold pad, and first gold pad includes at least the first antibody for having nano gold mark；Second gold pad includes at least the secondary antibody of nano gold mark；It include the third antibody of nano gold mark in first gold pad or the second gold pad；Control line, the first detection line, the second detection line and third detection line are disposed with along the opposite direction with sample flow on the basement membrane.The present invention provides the immuno-chromatographic test paper strips that can detect three kinds of mycotoxins simultaneously, reach the demand of simple, quick, high sensitivity on-site test, are base AFB1, tri- kinds of mycotoxins of T-2, DON quickly detect and the screening providing method of a large amount of sample.
Description
Technical field
The present invention relates to a kind of immunoassay test strip and preparation method, in particular to a kind of three mycotoxin colloidal golds
Immune chromatography test paper and preparation method.
Background technique
Mycotoxin (Mycotoxins) is the secondary metabolite for the low molecular weight that fungi generates during the growth process, extensively
It is general to be present in cereal and feed, seriously affect the health of humans and animals.The immune organ of mycotoxin main damage animal, in
Pivot nerve, liver, kidney and genitals official rank viscera tissue.In addition, some mycotoxins have very strong teratogenesis, carcinogenic, mutagenesis
Effect.Current known mycotoxin has more than 300 kinds, and endangering relatively serious mycotoxin to humans and animals has aspergillus flavus malicious
Plain B1(AFB1), vomitoxin (DON), zearalenone (ZEN), fumarubicin (FUM), ochratoxin A (OTA) with
And T-2 toxin etc..
Aflatoxin is one of strongest noxious material of toxicity known so far, in several aflatoxin,
Strongest toxicity is aflatoxin B1 (AFB1), and toxicity is higher than 10 times of potassium cyanide, belongs to special extremely toxic substance scope.Deoxidation snow
The main toxicity mechanism of rotten sickle-like bacteria enol (Deoxynivalenol, DON) be by conjunction with S60 ribosomes or inhibit peptidyl
The albumen of the activity suppression eukaryocyte of transferase synthesizes, and has acute toxicity, will lead to the weight of animals mitigation, and immunity is low
Under, also there is " three cause " effect, induce the harm such as liver canceration.T-2 toxin also belongs to the Trichothecenes of sickle-like bacteria generation
Toxin, main Fusarium sporotrichioides, the sickles such as Fusarium poae, and Fusarium acuminatum
The secretion of knife bacterium generates, T-2 acute toxicity cardinal symptom: vomiting, the long-term exposure of diarrhea, low dosage cause inflammatory reaction, are immunized
System disorders, animal food refusal, growth prevents, lymphoid tissue and intestinal mucosa are damaged, and animal may when searching for food these feeds
Take in a variety of mycotoxins, harm of the side effect that a variety of mycotoxins generate animal health and production performance than single toxin
Greatly.
In recent years, mycotoxin multiple detection method is quickly grown, and such as utilizes rare earth ion Eu3+And Sm3+As tracer
The Timed resolved fluoroimmunoassay (TRFIA) of foundation while to vomitoxin and zearalenone, it can be achieved that detect；
It detects a variety of mycotoxins in cereal simultaneously using liquid chromatography mass combination method (LC-MS/MS) and utilizes superelevation liquid phase color
Spectrum (UPLC) detects the composite pollution etc. of mycotoxin in coffee bean simultaneously.These method specificity are good, and sensitivity is higher, but grasp
Make complexity, takes a long time, be unable to satisfy the rapid screening of great amount of samples.Colloidal gold immunochromatographimethod technology is quick, simple due to having
Just, inexpensive feature, quickly grows in recent years, mainly includes competition law and double antibody sandwich method.Mycotoxin belongs to small point
Son, mostly uses competitive mechanism, and when sample to be examined feminine gender, detection line colour developing, when the sample to be examined positive, detection line does not develop the color.
Since colloid gold particle preparation is simple and labeling process is mild, had become before the Multiple detection type order based on immunochromatography
The research hotspot of field of detection of food safety.
Summary of the invention
Goal of the invention: convenient, fast and low meeting the present invention provides a kind of three colloidal gold immunochromatographimethod detection methods
While cost, realize to quickly being detected while above-mentioned three kinds of mycotoxins in cereal and feed.
Technical solution: a kind of three mycotoxins colloidal gold immune chromatography test of the present invention, including according to sample
The sequentially connected sample pad in flow direction, label pad, basement membrane and water absorption pad；The label pad includes the first gold pad and the second gold medal
Pad, first gold pad include at least the first antibody for having nano gold mark；Second gold pad includes at least nano gold mark
Secondary antibody；It include the third antibody of nano gold mark in first gold pad or the second gold pad；On the basement membrane along
The opposite direction of sample flow is disposed with control line, the first detection line, the second detection line and third detection line；Described
One detection line includes the first antigen with first antibody specific binding；Second detection line includes and secondary antibody is special
The second antigen that the opposite sex combines；The third detection line includes the third antigen in conjunction with third antibody specificity.
Preferably, the first antibody is AFB1 monoclonal antibody；The secondary antibody is T-2 monoclonal antibody, described
Third antibody is DON monoclonal antibody.
Preferably, the antigen of the first detection line scribing line is AFB1-BSA, concentration 0.2-0.6mgmL-1；Described
The antigen of two detection lines scribing line is T-2-BSA, concentration 0.4-1.2mgmL-1；The antigen of third detection line scribing line is
DON-BSA, concentration 0.4-1.2mgmL-1。
Preferably, between first detection line, the second detection line and third detection line between be divided into 2-3mm；The third
4-5mm is divided between detection line and control line.
Preferably, the partial size of the nanogold is 20-40nm.
The preparation method of above-mentioned three mycotoxins colloidal gold immune chromatography test, comprising the following steps: (1) preparation label
Pad: (1a) prepares colloidal nano gold solution: chlorauric acid solution being placed in magnetic agitation heater, when being heated to boiling, lemon is added
Lemon acid sodium solution, continuous heating is uniform and stable to solution colour, and continues to boil 5-10min, and room temperature natural cooling prepares nanometer
Goldc grains diameter is in 20-40nm；Antibody is added in colloidal nano gold solution in (1b), prepare nano gold mark first antibody solution,
The secondary antibody solution of nano gold mark and the third antibody-solutions of nano gold mark；(1c) will have been marked in step (1b)
Antibody-solutions are added dropwise in gold-labelled pad and dry, and prepare the first gold pad and the second gold pad；
(2) it prepares basement membrane: preparing the first detection line, the second antigen-BSA solution preparation second with the first antigen-BSA solution
Detection line, third antigen-BSA preparation third detection line, sheep anti-mouse igg solution prepare control line；
(3) by step (1) preparation the first gold pad and the second gold pad be sticked to form label pad, then, successively by water absorption pad,
Basement membrane, label pad and sample pad are bonded on bottom plate, are cut into strip, obtain three mycotoxin colloidal gold immune chromatography tests.
Preferably, in step (1b), the concentration of first antibody is 8-10 μ gmL in the first antibody solution-1, it is described
The concentration of secondary antibody solution is 8-10 μ gmL-1, the concentration of the third antibody-solutions is 6-10 μ gmL-1。
Preferably, in step (2), the antigen of the first detection line scribing line is AFB1-BSA, concentration 0.2-0.6mg
mL-1；The antigen of the second detection line scribing line is T-2-BSA, concentration 0.4-1.2mgmL-1；The third detection line is drawn
The antigen of line is DON-BSA, concentration 0.4-0.1.2mgmL-1。
Preferably, in step (2), the concentration of the sheep anti-mouse igg solution is 0.25-0.8mgmL-1。
Preferably, in step (2), 4-5mm is divided between the third detection line and control line.
The utility model has the advantages that (1) reaches the present invention provides the immuno-chromatographic test paper strip that can detect three kinds of mycotoxins simultaneously
Simply, quickly, the demand of the on-site test of high sensitivity, be base AFB1, tri- kinds of mycotoxins of T-2, DON quickly detect and big
Measure the screening providing method of sample；(2) label pad includes the first gold pad and the second gold pad in the present invention, and detection sensitivity increases,
Meanwhile multilayer gold pad slows down flow velocity while increasing reaction, increases the reaction time, improves the sensitivity of test strips.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of immune chromatography test paper of the present invention；
Fig. 2 is colloidal gold solution UV-Vis spectra scanning figure of the present invention；
Fig. 3 is colloidal gold transmission electron microscope image of the present invention；
Fig. 4 is the sensitivity test result figure of immunochromatography paper slip of the present invention.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawing.
Three mycotoxin colloidal gold immune chromatography test structures of the invention are as follows: as shown in Figure 1, pressing on bottom plate 1
Product flow direction is sequentially connected sample pad 2, label pad 3, basement membrane 4 and water absorption pad 5 by nitrocellulose film preparation in the same old way；Mark
Note pad 3 is made of the first gold pad 31 and the second gold pad 32, and there are three types of utilize nano gold mark for load altogether in label pad of the invention
Antibody, the first gold pad contains the first antibody of nano gold mark, and the second gold pad contains the secondary antibody of nano gold mark, nanometer
The third antibody of gold label can be located in the first gold pad or the second gold pad, and the first antibody in the present embodiment is anti-for AFB1 monoclonal
Body；Secondary antibody is T-2 monoclonal antibody, and third antibody is DON monoclonal antibody, and third antibody is located in the second gold-labelled pad.
Control line 41 is disposed with along the opposite direction with sample flow on basement membrane 4, the first detection line 42, second is examined
Survey line 43 and third detection line 44 contain the first antigen specifically bound with first antibody in first detection line；Second inspection
Survey line contains the second antigen with secondary antibody specific binding, and third detection line contains the in conjunction with third antibody specificity
Antigen iii is sheep anti-mouse igg solution in detection line, between the first detection line, the second detection line and third detection line between be divided into
3mm is divided into 4.5mm between third detection line and control line.
Three mycotoxin colloidal gold immune chromatography tests of the invention the preparation method is as follows:
1, material and instrument
1.1 instrument materials
Centrifuge, spectrophotometer, 3000 gold medal mark point sample instrument of XYZ, CM4000 cutting machine (Bio-dot company, the U.S.)；It is super
Pure water preparation instrument (the Millipore company in the U.S.)；GT000632 liquid chromatograph (Shimadzu Science and Technology Ltd., Japan)；Blood
Starch vortex mixer, pipettor.
1.2 reagent
T-2 toxin, vomitoxin (DON), AFB1 toxin, T-2 monoclonal antibody, DON monoclonal antibody, AFB1 monoclonal
Antibody (Sigma-Aldrich, the U.S.), sheep anti-mouse igg-HRP ELIAS secondary antibody (Nanjing Kai Moer biotechnology center), gold chloride
(Shanghai lark waffle Science and Technology Ltd.), boric acid (Nanjing Changji Bioisystech Co., Ltd), trisodium citrate (Nanjing base
Its Bioisystech Co., Ltd), ultrapure water, CN135 nitrocellulose membrane, water absorption pad, gold-labelled pad and sample pad (Shanghai Jie Ningsheng
Object), PEG (20000), PBS buffer solution (pH7.4 is purchased from Wuhan Biotechnology Co., Ltd, Google), acetonitrile and methanol are color
Spectrum level (Merck, Germany), sodium chloride, K2CO3Solution, (being purchased from Beijing Suo Laibao Science and Technology Ltd).
2, the preparation of three mycotoxin colloidal gold immune chromatography tests
The preparation and identification of 2.1 colloidal gold solutions
The chlorauric acid solution of 100mL 0.01% is placed in magnetic agitation heater, when being heated to boiling, is rapidly added 1% lemon
Lemon acid sodium solution 2.5mL prepares the colloidal gold solution of colloidal gold solution and 40nm that partial size is 20nm respectively, and continuous heating is to molten
Liquid color is uniform and stable, and continues to boil 5min, room temperature natural cooling, and ultrapure water supplies volume to 100mL.By ocular estimate and
Transmission electron microscopy identifies prepared colloid gold particle, select ultraviolet specrophotometer and Electronic Speculum to the colloidal gold of preparation into
Row identification.Select the sample that nanogold average grain diameter is 20nm as subsequent experimental, the spectrophotometer and Electronic Speculum knot of the sample
Fruit is as shown in Figures 2 and 3, it is seen that colloid gold particle size is more uniform.
The preparation of 2.2 nanometers of gold labeling antibodies
(1) determination of nano gold mark antibody Optimal pH
Respectively be added in 10 centrifuge tubes embodiment 1 preparation 1mL colloidal gold solution after be added 0,2,3,4,5,6,7,
8、9μL 0.2mol·L-1K2CO3, AFB1MAb antibody is diluted to 1 μ g. μ L-120 μ L (20 μ g) are taken to be added in 1-10 branch test tube,
5min is stood, room temperature reacts 20min in blood mixer.Every pipe adds the NaCl solution of 30 μ L10%, and concussion mixes, and room temperature is quiet
Only 2h visually observes the color of solution, without bluish violet precipitating, K2CO3Solution adds the corresponding pH body of least test tube colloidal gold
System.It marks the pH optimization method of T-2mAb, DON mAb reaction with AFB1 mAb, is added in the colloidal gold solution of final choice 1mL
2μL 0.2M K2CO3For solution as Optimal pH, observation color is claret, and centrifugation colloidal gold is not assembled like drift sand.
(2) nanogold particle antibody labelled amount measures
The determination of minimum mark amount: 2mL centrifuge tube 10 are taken, is separately added into and is adjusted to Optimal pH with solution of potassium carbonate
Different amounts of 1,5,10,15,20,25,30,35,40,0 μ g of antibody purification is added, gently in colloidal gold solution 1mL into each pipe according to this
It is light to mix, after reacting at room temperature 30min, after the NaCl of 0.1mL 10% is added in above-mentioned test tube, see within mixing static 2 hours or more
Examine result.Not plus antibody and be added the insufficient test tube of amount of antibody will appear it is red become blue coagulation phenomenon, and amount of antibody is added and reaches
Or more than the test tube of minimum stable quantity then colloidal gold red it is constant.It is wherein every milliliter of colloidal gold containing the minimum test tube of antibody
The necessary amount of antibody of stable state.Increased on this basis to reach actual needs, the three kinds of mycotoxins finally measured are anti-
Body AFB1MAb, DON mAb, T-2mAb minimum stable labelling amount are respectively 5 μ gmL-1、5μg·mL-1、5μg·mL-1。
Optimum mark concentration determines: taking 1.5mL centrifuge tube 9, is separately added into and is adjusted to Optimal pH with solution of potassium carbonate
Colloidal gold solution 1mL, not same amount antibody-solutions are successively added into each pipe, until antibody final concentration is slightly lower in solution, be equal to, high
In antibody minimum stable labelling concentration.Antibody final concentration is respectively x-3 μ g, x-2 μ g, x-1 μ g, x μ g, x+1 μ g, x+2 in 1-7 pipe
μ g, x+3 μ g antibody/mL nano-Au solution, x are minimum mark amount, and ultrapure water (control group) is added in 8-9 branch test tube.It is slight to shake
It is put into blood plasma vortex mixer after dynamic and reacts 20min, 10% (m/v) BSA solution, 50 μ L is added, is put into blood plasma vortex mixer and reacts
20min is added 5%PEG (20000), after stirring 17min, 4 DEG C of static 2h, by color there is no changing, without colloidal gold
The test tube of aggregation is centrifuged.It will be put into a centrifuge equipped with the colloidal gold test tube marked, 11000r is centrifuged 20min, will manage
In supernatant slowly drawn out and discarded with pipettor, then draw 500 μ L redissolve liquid the colloid gold label being concentrated is resuspended
Antibody-solutions, 4 DEG C of storages.The gold solution that 10 μ L different antibodies concentration markers are taken with pipettor, is uniformly laid in the gold of blank
On mark pad, 15min observes color change.Getting colors, obvious, the smallest one group of antibody usage amount as colloidal gold solution antibody
Label dosage, the three kinds of mycotoxin antibody A FB finally measured1MAb, DON mAb, T-2 mAb optimum mark concentration difference
For 8 μ gmL-1、8μg·mL-1、6μg·mL-1。
The determination of 2.3 detection line antigens and control line secondary antibody peridium concentration
By AFB1- BSA is according to 0.2mgmL-1、0.4mg·mL-1、0.6mg·mL-1Concentration be sprayed on nitrocellulose membrane
As detection line (T line), DON-BSA and T-2-BSA is respectively according to 0.4mgmL-1、0.8mg·mL-1、1.2mg·mL-1It is dense
Degree is sprayed on nitrocellulose membrane as detection line (T line), after dry, is added drop-wise to blank test strips gold with the antibody-solutions marked
On mark pad, drying.It is added after 100 μ L PBS and observes the color of each test strips after 10min.Select that color is obvious, envelope antigen
The minimum concentration of concentration is as best T line concentration.
Sheep anti-mouse igg is selected into 0.3mgmL-1、0.6mg·mL-1、0.8mg·mL-1、1mg·mL-1Concentration be sprayed onto nitre
It is used as control line (C line) on sour tunica fibrosa, after dry, is added drop-wise in blank test strips gold-labelled pad with the antibody-solutions marked,
Drying.It is added after 100 μ L PBS and observes the color of each test strips after 10min.Selection color is obvious, coating secondary antibody concentration is minimum
Concentration as best C line concentration.
By testing above, AFB1-BSA, T-2-BSA, DON-BSA antigen coat amount are respectively 0.4mgmL-1、
0.4mg·mL-1、0.8mg·mL-1.The final final package amount for determining three kinds of toxin coating secondary antibodies is 0.25mgmL-1。
The preparation of 2.4 test strips
(1) preparation of basement membrane: by the good detection line of above-mentioned optimization and control line concentration, by AFB1- BSA, T-2-BSA are complete
Antigen, which is used, contains 0.16% glycerol, and the phosphate buffer solution that pH is 5 is diluted to 0.4mgmL-1, buffered with the PBS that pH is 7.4
DON-BSA is diluted to 0.8mgmL by solution-1, nature controlling line antibody is diluted to 0.3mgmL-1.When coating according on to
Under sequence by the first detection line AFB1- BSA, the second detection line T-2-BSA, third detection line DON-BSA are successively coated on nitre
It is used as detection line (T line) on acid cellulose film (Nitrocellulose Membrane, NC), controls wire spraying sheep anti-mouse igg,
The spacing of control line and the first detection line is 4.5mm, and spacing is between the first detection line, the second detection line and third detection line
The quantity for spray of 3mm, detection line and nature controlling line is 17 μ L/cm, and film is placed on 65 DEG C of drying box drying 1h, kept dry after the completion.
(2) it the preparation of label pad: prepares containing 1%PVPK30 dimension ketone, 1% sucrose, 0.5% Tween-20,1%BSA
0.1mol.L-1The confining liquid of PBS is put into 37 DEG C of drying box drying, by marking nano by glass fibre membrane infiltration after confining liquid
Au probe solution (1:10 dilution) uniformly infiltrates on 8964 glass fibre membranes of 3mm × 420mm, the monoclonal that will be prepared
Antibody combines on the glass fibers, and drying at room temperature obtains gold-labelled pad: being specially the glass for the gold solution paving for marking T-2, DON
Fiber is the second gold pad, and the glass fibre of AFB1 colloidal gold solution paving is the first gold-labelled pad.
(3) assembling of test strips: sample pad, gold-labelled pad, basement membrane and water absorption pad are assembled, on bottom plate 1, according to sample
The sequentially connected sample pad 2 in product flow direction, label pad 3, basement membrane 4 and water absorption pad 5.Label pad 3 is by the first gold pad 31 and
Two gold pads 32 press, and label pad size is width 3mm, long 4mm, on basement membrane 4 successively along the opposite direction with sample flow
It is provided with control line 41, the first detection line 42, the second detection line 43 and third detection line 44, is cut into 4mm's wide with cutting machine
Film is placed on 65 DEG C of drying box drying 1h after the completion, obtains test strips of the invention by immuno-chromatographic test paper strip to detect.
AFB1, T-2, DON immuno-chromatographic test paper strip detect three kinds of toxin using A competitive inhibition method, when three kinds in test sample
The content of any one toxin of toxin be greater than or equal to detection line it is offline when, the mAb- colloidal gold in gold-labelled pad will be with sample
Toxin in product combines, and the gold labeling antibody binding site in gold-labelled pad is closed, and can not be combined with the toxin-BSA in detection line,
There is not red in detection line, therefore is known as the line that disappears, and test sample is the positive.When content of toxins is lower than detecting offline in sample, gold
Antigen binding site is specifically bound with the toxin-BSA in detection line in labeling antibody, and red occurs in detection line, and sample is
It is negative.
Influence of the content of methanol to test strips in 2.5 feed extracts
In mycotoxin detection, methanol, acetonitrile of high concentration etc. are commonly used in sample in the extraction process of mycotoxin
Organic solvent, and immunochromatography reaction in antibody activity will receive high concentration organic solvent influence, to cause test paper
The colour generation of detection line and nature controlling line, therefore, in detection process, sample extracting solution needs organic to reduce by dilution appropriate
The concentration of solvent carries out immunochromatographiassays assays under the premise of not influencing antibody activity.Colloidal gold strip is inserted into different first
In the sample solution of determining alcohol (0,10,20,30,40,50,60,70,80%) observation test strips develop the color feelings after 15min at room temperature
Condition, the methanol concentration that choosing test strips reaction can normally be resistant to are used for sample pre-treatments, as a result, it has been found that, when methanol is more than 30%
When T line and the colour developing of C line it is fuzzy, the content for reducing methanol can optimize the color developing effect of immuno-chromatographic test paper strip, finally select first
Determining alcohol 20% is optimum concentration.
2.6 immuno-chromatographic test paper strip performance detections
(1) sensitivity technique
It after DON, AFB1 and T-2 negative sample detected through efficient liquid phase drying, is ground up, sieved, adds respectively by table 1
After entering toxin standard solution and mixing well, left at room temperature over night is to be checked.
The additive amount of toxin in 1 sample of table
It prepares feed extract: weighing sample that 1g is ground into 10mL centrifuge tube, 5mL 20% (v/v) methanol-water is added
Supernatant after vortex oscillation 5min, is crossed quantitative filter paper by solution, and 1mL 30mmol PBST is added in supernatant solution after taking 1mL to filter
Buffer solution dilution, takes the feed extract to be added in test strips sample pad, measures the line concentration that disappears, the results showed that, work as feed
AFB in extracting solution1, DON and T-2 concentration is when being respectively 2,100,20ng/mL, three detections line color substantially completely disappears, i.e.,
It is test strips to AFB1, DON and the macroscopic line concentration that disappears of T-2, the corresponding forage poisoning element line concentration that disappears is respectively AFB1
20μg.kg-1、T-2 0.2mg.kg-1、DON 1mg.kg-1。
(2) detection of the accuracy, reproducibility, stability, specificity of immuno-chromatographic test paper strip
Specificity analysis: toxin, which is diluted to, with 20% methanol aqueous solution keeps the single toxin of 3 times of concentration of T heading line off molten
Liquid, every test tube take 70 μ L that sample pad is added.At room temperature after 15min, observation colour developing result.
The result shows that: as shown in Figure 4, wherein (a) is negative control, (b) is to contain toxin AFB1Solution detection knot
Fruit, (c) be the solution containing toxin T-2 testing result, (d) be the solution containing toxin DON testing result, (e) for containing
There are three types of the testing results of the mixed solution of toxin, it can be seen from the figure that three joint-trial paper slips detect single mycotoxin solution,
Due to competitive reaction, corresponding detection line does not develop the color, and the corresponding detection line colour developing of other mycotoxins is suitable with negative control.It is added
The test strips of three kinds of mycotoxin mixed solutions make since with corresponding gold labeling antibody competitive reaction occurs for three kinds of mycotoxins
Obtaining antibody can not be integrated in detection line, therefore three detections line does not develop the color.
Stability, reproducibility and accuracy measurement: the critical line concentration measured according to 2.6 first parts is respectively set three
The mycotoxin mixed solution of a quantitative concentrations, first is AFB1, T-2 and DON concentration be lower than critical line concentration, second
For AFB1, T-2 and DON concentration be equal to critical line concentration, third is AFB1, T-2 and DON concentration be higher than critical line concentration,
The mycotoxin mixed solution of quantitative concentrations is added into blank Feed Sample extracting solution, each concentration sets 20 repetitions, observation
And statistic mixed-state is as a result, with the accuracy of analysis test paper item.In the analysis of test strips reproducibility, exempt to same batch and between criticizing
Epidemic disease chromatograph test strip observes the difference of 10 replications, the consistency of comparative analysis measurement result.It is right in estimation of stability
The same a collection of test strips of preparation are placed 2 weeks at 37 DEG C, wherein taking out test strips daily, observation test strips colour developing situation compares detection
Colour developing result when analysis.
The result shows that: when mycotoxin addition concentration is low compared with test strips detection limit concentration, test strips detection line and feminine gender are right
It is suitable according to colour developing；When concentration of mycotoxins detects between limit and the line value that disappears between test strips, the colour developing of test strips detection line is obvious
It shoals；When concentration of mycotoxins disappears line value higher than test strips, detection line does not develop the color.Have in replication 20 times 1 time and ties
Fruit is abnormal, and the line that do not occur disappearing is in false negative, accuracy rate 95%.Illustrate that accuracy and the reproducibility of this immuno-chromatographic test paper strip are equal
Preferably.
In 37 DEG C of acceleration for stabilization tests, product is placed 1 week at 37 DEG C to be equal to room temperature 6 months；37 DEG C are placed 2 weeks quite
In room temperature 12 months.Packaged test strips are placed in 37 DEG C in experiment to place 2 weeks, wherein taking out test strips daily, are seen
Examine test strips colour developing situation.The detection line of test strips is still clear after 10 days, and the colour developing of test strips detection line starts to become at 12-14 days
Shallowly, but still there is apparent competitive inhibition reaction, therefore effective service life of test strips can be set to 10 months or more.
2.7 colloidal gold immuno-chromatography test paper strips try compared with HPLC
The 10 parts of Feed Samples extracted out at random are detected simultaneously with HPLC inspection, are prepared feed extract according to 2.6 parts, are detected
AFB in feed extract as the result is shown1Toxin is more than 2ngmL-1T line thoroughly disappears, as slightly below 2ngmL-1When T heading line off
It is not thorough；T-2 is more than 20ngmL-1T line thoroughly disappears；DON is more than 100ngmL-1T line thoroughly disappears, immune chromatography test paper
The testing result and liquid phase testing result match rate of item are 100%.
Claims (10)
1. a kind of three mycotoxin colloidal gold immune chromatography tests, which is characterized in that including according to sample flow direction successively
Sample pad, label pad, basement membrane and the water absorption pad of connection；The label pad includes the first gold pad and the second gold pad, and described first
Gold pad includes at least the first antibody for having nano gold mark；Second gold pad includes at least the secondary antibody of nano gold mark；
It include the third antibody of nano gold mark in first gold pad or the second gold pad；On the basement membrane along with sample flow
Opposite direction is disposed with control line, the first detection line, the second detection line and third detection line；The first detection line packet
Contain the first antigen specifically bound with first antibody；Second detection line includes to specifically bind with secondary antibody
Second antigen；The third detection line includes the third antigen in conjunction with third antibody specificity.
2. three mycotoxins colloidal gold immune chromatography test according to claim 1, which is characterized in that described first is anti-
Body is AFB1 monoclonal antibody；The secondary antibody is T-2 monoclonal antibody, and the third antibody is DON monoclonal antibody.
3. three mycotoxins colloidal gold immune chromatography test according to claim 2, which is characterized in that first inspection
The antigen of survey line scribing line is AFB1-BSA, concentration 0.2-0.6mgmL-1；The antigen of the second detection line scribing line is T-2-
BSA, concentration 0.4-1.2mgmL-1；The antigen of the third detection line scribing line is DON-BSA, concentration 0.4-1.2mg
mL-1。
4. three mycotoxins colloidal gold immune chromatography test according to claim 1, which is characterized in that first inspection
2-3mm is divided between survey line, the second detection line and third detection line；It is divided between the third detection line and control line
4-5mm。
5. three mycotoxins colloidal gold immune chromatography test according to claim 1, which is characterized in that the nanogold
Partial size be 20-40nm.
6. a kind of preparation method of three mycotoxins colloidal gold immune chromatography test a method as claimed in any one of claims 1 to 5,
It is characterized in that, comprising the following steps:
(1) label pad is prepared:
(1a) prepares colloidal nano gold solution: chlorauric acid solution being placed in magnetic agitation heater, when being heated to boiling, lemon is added
Lemon acid sodium solution, continuous heating is uniform and stable to solution colour, and continues to boil 5-10min, and room temperature natural cooling prepares nanometer
Goldc grains diameter is in 20-40nm；
Antibody is added in colloidal nano gold solution in (1b), prepares the first antibody solution of nano gold mark, nano gold mark
The third antibody-solutions of secondary antibody solution and nano gold mark；
The antibody-solutions marked in step (1b) are added dropwise in gold-labelled pad and dry by (1c), prepare the first gold pad and the second gold medal
Pad；
(2) it prepares basement membrane: preparing the first detection line, the second detection of the second antigen-BSA solution preparation with the first antigen-BSA solution
Line, third antigen-BSA preparation third detection line, sheep anti-mouse igg solution prepare control line；
(3) first gold pad of step (1) preparation and the second gold pad are sticked to form label pad, then, successively by water absorption pad, base
Film, label pad and sample pad are bonded on bottom plate, are cut into strip, obtain three mycotoxin colloidal gold immune chromatography tests.
7. the preparation method of three mycotoxins colloidal gold immune chromatography test according to claim 6, it is characterised in that
In step (1b), the concentration of first antibody is 8-10 μ gmL in the first antibody solution-1, the secondary antibody solution it is dense
Degree is 8-10 μ gmL-1, the concentration of the third antibody-solutions is 6-10 μ gmL-1。
8. the preparation method of three mycotoxins colloidal gold immune chromatography test according to claim 6, it is characterised in that
In step (2), the antigen of the first detection line scribing line is AFB1-BSA, concentration 0.2-0.6mgmL-1；Second inspection
The antigen of survey line scribing line is T-2-BSA, concentration 0.4-1.2mgmL-1；The antigen of the third detection line scribing line is DON-
BSA, concentration 0.4-0.1.2mgmL-1。
9. the preparation method of three mycotoxins colloidal gold immune chromatography test according to claim 6, it is characterised in that
In step (2), the concentration of the sheep anti-mouse igg solution is 0.25-0.8mgmL-1。
10. the preparation method of three mycotoxins colloidal gold immune chromatography test according to claim 6, it is characterised in that
In step (2), 4-5mm is divided between the third detection line and control line.
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