CN107589265A - A kind of time-resolved fluorescence test strips for detecting aflatoxin B1 and its application - Google Patents
A kind of time-resolved fluorescence test strips for detecting aflatoxin B1 and its application Download PDFInfo
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- CN107589265A CN107589265A CN201710753370.6A CN201710753370A CN107589265A CN 107589265 A CN107589265 A CN 107589265A CN 201710753370 A CN201710753370 A CN 201710753370A CN 107589265 A CN107589265 A CN 107589265A
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- aflatoxin
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- resolved fluorescence
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- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title claims abstract description 81
- 239000002115 aflatoxin B1 Substances 0.000 title claims abstract description 80
- 229930020125 aflatoxin-B1 Natural products 0.000 title claims abstract description 80
- 238000012360 testing method Methods 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 239000011248 coating agent Substances 0.000 claims abstract description 16
- 238000000576 coating method Methods 0.000 claims abstract description 16
- 239000000427 antigen Substances 0.000 claims abstract description 15
- 102000036639 antigens Human genes 0.000 claims abstract description 15
- 108091007433 antigens Proteins 0.000 claims abstract description 15
- 238000010521 absorption reaction Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 235000017060 Arachis glabrata Nutrition 0.000 claims abstract description 10
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- 230000000274 adsorptive effect Effects 0.000 claims abstract description 10
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- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 claims description 14
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- 238000003756 stirring Methods 0.000 claims description 10
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- PCNFLKVWBDNNOW-UHFFFAOYSA-N 4-hydrazinylbenzoic acid Chemical class NNC1=CC=C(C(O)=O)C=C1 PCNFLKVWBDNNOW-UHFFFAOYSA-N 0.000 claims description 6
- 241000228197 Aspergillus flavus Species 0.000 claims description 5
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- JKKCSFJSULZNDN-UHFFFAOYSA-N gonyautoxin v Chemical compound N=C1NC(COC(=O)NS(O)(=O)=O)C2NC(=N)NC22C(O)(O)CCN21 JKKCSFJSULZNDN-UHFFFAOYSA-N 0.000 claims description 4
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- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of time-resolved fluorescence test strips for detecting aflatoxin B1 and its application.Test strips include sample absorption pad, reaction film and adsorptive pads, and reaction film is overlapped in reaction film left and right ends respectively positioned at centre, sample absorption pad with adsorptive pads；Sample absorption pad is provided with sample application zone and microballoon area, and microballoon area is loaded with aflatoxin B1 time time-resolved fluorescence microballoon；Reaction film is provided with detection line (T lines) and nature controlling line (C lines), and T lines connect coating aflatoxin B1 antigen (coating antigen), C lines coating anti-rabbit antibody.Present invention also offers the method that aflatoxin B1 in the samples such as a kind of above-mentioned aflatoxin B1 ELISA test strip grain of application, edible oil, peanut, corn class feed (premix) remains.Test strips provided by the present invention have the characteristics that simple to operate, high sensitivity, quantitative detection, quick detection, cost are low, are adapted to the examination and on-site supervision of great amount of samples.
Description
Technical field
The present invention relates to a kind of time-resolved fluorescence test strips for detecting aflatoxin B1 and its application, belong to the time point
Fluoroimmunoassay (TRFIA) technical field is distinguished, for samples such as grain, edible oil, peanut, corn class feeds (premix)
The content detection of aflatoxin B1 in product.
Background technology
Aflatoxin B1 is the derivative of dihydrofuran cumarin, is that carcinogenicity is most strong in known chemical substance
It is a kind of.Aflatoxin B1 has strong toxicity to humans and animals, and its toxic action is mainly the infringement to liver.Aspergillus flavus
The food of toxin B1 pollutions is mainly the oil and foodstuffses such as peanut, corn, paddy, wheat, peanut oil, and with southern high-temperature, high humidity
Area is contaminated the most serious.Aflatoxin is heat-resisting, 280 DEG C of ability cleavables, therefore typically cooks and be difficult to destroy under processing temperature.
The toxicity of aflatoxin B1 than vomitoxin 30 times of strong toxicity, than 20 times of the strong toxicity of zearalenone.Aspergillus flavus
Toxin B1 acute toxicity is 10 times of potassium cyanide, and 68 times of arsenic, chronic toxicity can induce canceration, and carciongenic potency is dimethyl
75 times of nitrosamine, the primary carcinoma of liver of people are also likely to relevant with aflatoxin.Therefore, correlation quality testing department in China's is to this
Material has carried out limitation regulation.
At present, detecting the method for aflatoxin B1 has thin-layered chromatography, liquid chromatography, ELISA, liquid phase color
Compose tandem mass spectrometry etc..The accuracy of instrument detection method is higher, but expensive equipment is, it is necessary to technical professional, detection
Step is complicated, it is difficult to batch detection sample, is not suitable for laboratories batch, quick detection sample.And immunology detection side
Method is simple to operate, quick, sensitive, can detect most samples simultaneously, is preferable quick screening means.
The content of the invention
It is an object of the invention to provide a kind of high sensitivity, the aflatoxin that simple to operate, cost is low, detection time is short
B1 time-resolved fluorescence test strips.
A kind of aflatoxin B1 time-resolved fluorescence test strips provided by the present invention, the test strips include sample and absorbed
Pad, reaction film and adsorptive pads three parts.Reaction film is overlapped in a reaction film left side respectively positioned at centre, sample absorption pad with adsorptive pads
Right both ends.Wherein, sample absorption pad is provided with sample application zone and microballoon area, and microballoon area is loaded with time-resolved fluorescence microballoon；Reaction
Film is provided with detection line (T lines) and nature controlling line (C lines), and T lines connect coating aflatoxin B1 antigen, C lines coating anti-rabbit antibody.
The aflatoxin B1 time-resolved fluorescence microballoon, including detection microballoon and Quality Control microballoon, detection microballoon are table
Face is coated with the fluorescent microsphere of aflatoxin B1 monoclonal antibody, and Quality Control microballoon is that pan coating has the glimmering of rabbit-anti labelled protein
Light microballoon.
Bright-coloured series elements compound is filled with the fluorescent microsphere；Preferably, the bright-coloured system member rope huge legendary turtle compound is europium chelant thing；
Optimal, the europium chelant thing can be Eu (TTA) 3/TOPO or Eu (TTA) 3/Phen.
The albumen can be cow's serum Y- globulin (BGG) or bSA (BSA).
The time-resolved fluorescence microspherulite diameter scope is 100-1000nm.
On the nitrocellulose filter, coated antibody is aflatoxin B1 antigen on T lines, coated anti-rabbit on C lines
Antibody.
The aflatoxin B1 time-resolved fluoroimmunoassay quantitative testing test paper bar bottom is provided with plastic bottom board.
It is to be obtained by aflatoxin B1 haptens with carrier protein couplet that the T lines, which connect coating aflatoxin B1 antigen,
Arrive, the carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
The aflatoxin B1 monoclonal antibody is using aflatoxin B1 hapten-carrier protein conjugate as exempting from
Epidemic focus prepares, and is to be secreted to obtain by the strain of aflatoxin B1 monoclonal antibody hybridoma cell；The anti-rabbit antibody be by
Antibody mediated immunity sheep obtains in rabbit source.
The sample absorption pad is Fusion5 films or other function identical films；The conjugate release pad can be glass
Cotton or polyester material；The adsorptive pads are adsorptive pads；The reaction film can be nitrocellulose filter or cellulose acetate film.
It is a further object to provide a kind of method for preparing above-mentioned test strips, it includes step：
(1) prepared by Quality Control microballoon：
1. use biotinylated protein；
2. aldehyde group modified fluorescent microsphere is coated with using above-mentioned labelled protein；
(2) prepared by haptens：Aflatoxin B1 and 4- hydrazino-benzoic acids are reacted to obtain aflatoxin B1 haptens
Product；
(3) by aflatoxin B1 haptens and carrier protein couplet, aflatoxin B1 hapten-carrier albumen is obtained
Conjugate；
(4) new zealand white rabbit is immunized with aflatoxin B1 hapten-carrier protein conjugate, by new zealand white rabbit
Splenocyte and myeloma cell obtain aflatoxin B1 monoclonal hybridoma strain by merging, screening；
(5) new zealand white rabbit IgG immune health goats are extracted, obtain anti-rabbit antibody；
(6) microballoon is detected to prepare：Aldehyde group modified fluorescent microsphere is coated with using the monoclonal antibody of aflatoxin B1；
(7) blank kilocalorie is pasted：Nitrocellulose filter is pasted onto among plastic bottom board, Fusion5 films and adsorptive pads point
Nitrocellulose filter left and right ends are not overlapped in；
(8) film is sprayed：The fluorescent microsphere of the fluorescent microsphere of rabbit-anti labelled protein and aflatoxin B1 monoclonal antibody is adopted
With release buffer solution mixed diluting to finite concentration, the microballoon area of Fusion5 films is sprayed onto；Aflatoxin B1 antigen and anti-rabbit resist
After body dilution, the T lines and C line positions of nitrocellulose filter are sprayed onto respectively；
(9) dry and cut：By the above-mentioned kilocalorie drying for having sprayed reagent, slitting.
The release buffer solution used in spray film step contains 10%-20% sucrose, 3%-10% trehaloses, 0.5%-1%
The double trimethylsilyl acetamides (BSA) of N, 0-, 0.2%-0.5% gentamicins.
The final concentration of 0.5mg/mL-2mg/mL of microballoon, Quality Control microballoon final concentration 0.05mg/ are detected after spraying the dilution of film step
mL-0.2mg/mL；T line antigen final concentration 0.5mg/mL-2mg/mL, C line antigen final concentrations 0.5mg/mL-2mg/mL；C, T line spray
Film liquid amount is 0.5 μ L/cm-1.5 μ L/cm, and microballoon spray film amount is 2 μ L/cm-8 μ L/cm.
The drying dried and cut described in step can be in constant temperature oven or drying room, 37 DEG C of drying 8-12 hours.
It is a further object to provide the above-mentioned ELISA test strip grain of one kind application, edible oil, peanut, jade
The method that aflatoxin B1 remains in the samples such as rice class feed (premix), it includes step：
(1) sample pre-treatments；
(2) detected with test strips；
(3) testing result is analyzed.
The present invention aflatoxin B1 time-resolved fluorescence test strips using high degree of specificity antibody antigen reaction and
Immunochromatographiassays assays technology, aflatoxin B1 monoclonal antibody-time-resolved fluorescence label is fixed on Fusion5 films
On, the aflatoxin B1 in sample in flow process, with Fusion5 films aflatoxin B1 monoclonal antibody-when
Between resolved fluorometric label combine, formed drug-antibody-time-resolved fluorescence label.Medicine in sample is examined with reaction film
Aflatoxin B1 hapten-carrier protein conjugate competition binding aflatoxin B1 monoclonal antibody-time on survey line
Resolved fluorometric label, final application immunochromatography detector calculate the aflatoxin B1 residual contained in analyte sample fluid
Amount.
The test strips of the present invention have that high specificity, high sensitivity, quantitative detection, cost be low, simple to operate, detection time
The advantages of short, suitable various units use, storage is simple, long shelf-life.It is residual with ELISA test strip aflatoxin B1 of the present invention
The method stayed is easy, quick, directly perceived, accurate, applied widely, cost is low, easily promotes the use of.
Brief description of the drawings
Fig. 1 is aflatoxin B1 hapten synthesis route map.
Fig. 2 is aflatoxin B1 haptens hydrogen nuclear magnetic resonance spectrogram.
Fig. 3 is aflatoxin B1 coating antigen/immunogene synthetic route chart.
Embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.
The preparation of the aflatoxin B1 test strip of embodiment 1
The preparation method of the test strips mainly includes the following steps that：
1) the sample absorption pad for being coated with aflatoxin B1 monoclonal antibody-time-resolved fluorescence label is prepared；
2) prepare with the detection line for being coated with aflatoxin B1 hapten-carrier protein conjugate and be coated with anti-rabbit
The reaction film of the nature controlling line of antibody；
3) test strips 1) will be assembled into sample absorption pad, reaction film and the adsorptive pads 2) prepared.
Substep narration in detail below：
1st, the preparation of aflatoxin B1 haptens
31.2mg aflatoxin B1s are dissolved in 2ml ethanol, add 12mg sodium cyanoborohydrides, 0 DEG C of stirring and dissolving.Add
Enter 16.5mg 4- hydrazino-benzoic acids, stir, be to slowly warm up to 50 DEG C, stir 1h.Solvent evaporated, column chromatography purifying, it is white to obtain 39mg
Color solid, yield 88%.The carboxyl peak of the methyl peak of 3.9 aflatoxin B1 and 12.7 4- hydrazino-benzoic acids is present,
Illustrate hapten synthesis success.Haptens prepared by this method is to use 4- hydrazino-benzoic acids, and carboxyl is introduced by single step reaction, and
And linking arm is 6 keys, than 3 keys of connection brachium of CMO methods commonly used in the art, make haptens with after carrier protein couplet more
Easily exposed to outer, enhancing immune response.
2nd, the preparation of immunogene
133mg BSA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Aflatoxin B1 haptens and BSA ratios can be
Adjusted in the range of 1: 1~60: 1, the application selection add 44.8mg aflatoxin B1s haptens (with BSA mol ratios for
50: 1), add EDC hydrochloride 10mg, 2h is stirred at room temperature.The 48h that dialyses is moved into bag filter.Aflatoxin is obtained under the ratio
B1 immunogenes can expose more aflatoxin B1 haptens in antigenic surface, so as to preferably cause for aspergillus flavus
Toxin B1 immune response.
3rd, the preparation of coating antigen
86mg OVA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Aflatoxin B1 haptens and OVA ratios can be 1
: adjusted in the range of 1~40: 1, the application selection (is 40 with OVA mol ratios adding 35.8mg aflatoxin B1s haptens
: 1), EDC hydrochloride 10mg are added, 2h is stirred at room temperature.The 48h that dialyses is moved into bag filter.Aflatoxin B1 is obtained under the ratio
Coating antigen, can fully and antibody response when with antigenic competition antibody in sample, also will not be because of bag so as to avoid false positive
It is excessive by former exposed sites, steric hindrance is formed, so as to avoid false negative.
4th, the preparation of aflatoxin B1 monoclonal antibody
Immunogene is injected in Balb/c new zealand white rabbit bodies, produces antiserum.Take immune Balb/c New Zealand great Bai
Rabbit splenocyte merges with SP2/0 myeloma cell, screens positive hole, obtains the hybridoma of stably excreting monoclonal antibody
Strain.Cell suspension is made with frozen stock solution in hybridoma, it is standby.Hybridoma is placed in cell culture medium, at 37 DEG C
Under the conditions of cultivated, obtained nutrient solution is purified with octanoic acid-saturated ammonium sulfate method, obtains monoclonal antibody, -20 DEG C
Preserve.
The cell culture medium is that calf serum and sodium acid carbonate are added into RPMI1640 culture mediums, calf serum is existed
Final concentration of 20% (mass fraction) in cell culture medium, final concentration of 0.2% (matter of the sodium acid carbonate in cell culture medium
Measure fraction)；The pH of the cell culture medium is 7.4.It is immunogene to pathogen-free domestic using rabbit source antibody using sheep as immune animal
Sheep is immunized, and obtains anti-rabbit antibody.
5th, the preparation of microspheres solution, detection line (T lines) solution and nature controlling line (C lines) solution
(1) configuration of microspheres solution
With release buffer solution (containing 20% sucrose, 5% trehalose, 0.5%BSA, 0.2% gentamicin) by Quality Control microballoon
Time-resolved fluorescence microballoon mixed liquor, the final concentration of 0.1mg/mL-0.3mg/mL of Quality Control microballoon, detection are configured to detection microballoon
The final concentration of 0.8mg/mL-1.2mg/mL of microballoon.
(2) preparation of detection line (T lines) solution
Aflatoxin B1 antigenic compound is diluted to 0.4mg/mL-0.6mg/mL with 0.01M PB solution.
(3) preparation of nature controlling line (C lines) solution
Streptavidin is diluted to 0.4mg/mL-0.6mg/mL with 0.01M PB solution.
6th, the preparation of test strips
Reaction film is overlapped in reaction film left and right ends with blotting paper, is pasted onto and carries respectively positioned at centre, sample absorption pad
On the plastic bottom board of gum.C, T line spray film liquid amount are 0.6 μ L/cm-1.2 μ L/cm, and microspheres solution spray film amount is 2 μ L/cm-6 μ L/
cm.The aflatoxin B1 for having sprayed reagent is stuck in greatly in constant temperature oven into 37 DEG C to dry 12 hours.By the aflatoxin of drying
B1 kilocalories cut into the paper slip of 3mm-5mm width, that is, obtain aflatoxin B1 test strips.
The detection that aflatoxin B1 remains in the sample of embodiment 2
1st, sample treatment
Sample diluting liquid is prepared：With deionized water by 10X sample diluting liquids (pH value 7.0,1mol/L phosphate-buffered
Liquid) be diluted by 1: 9 volume ratio, i.e., 1 part of 10X sample diluting liquid adds 9 parts of deionized waters.
Sample extracting solution is prepared：1.5g NaCl accurately are weighed in 50ml centrifuge tubes, and it is fully molten to add 25ml deionized waters
Solution adds the mixing of 25ml methanol solutions, standby.
It is accurate to weigh the samples such as grain/edible oil/peanut/corn class feed (premix) after 10.0g is uniformly crushed
Product (or measuring 10ml liquid samples) are in 50ml centrifuge tubes, then accurate addition 20ml sample extracting solutions, will centrifuge lid lid
Tightening seal, 5min is fully vibrated, stand or centrifuge 2min in 4000rpm, obtain supernatant (if sample, which is edible oil, takes subnatant)；
Supernatant (or subnatant) is suctioned out into 1.0ml in 50ml centrifuge tubes with suction pipe, the sample diluting liquid for adding 4ml mixes,
It is to be checked.
2nd, detected with test strips
(1) test strips and extract solution are recovered to room temperature.
(2) 60 μ L measuring samples are accurately drawn with pipettor, (being careful not to suck bubble during sampling) is vertically slowly added dropwise
In well (S), start timing after sample-adding, read result within 8 minutes after sample-adding.
(3) reagent card is inserted in the carrier of immunochromatography detector, presses detection key, instrument will stick into test automatically
Row scanning.
(4) testing result is read from the display screen of immunochromatography detector.
3rd, testing result is analyzed
Positive cutoff value：≥5ng/ml.
The sample detection example of embodiment 3
1st, positive cutoff value is tested
The samples such as blank grain/edible oil/peanut/corn class feed (premix) are taken, add aspergillus flavus poison respectively
Plain B1 takes test paper to final concentration of 2.5ng/ml, 5ng/ml, 7.5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml
Bar is detected, and each sample is repeated three times.
During with samples such as ELISA test strip grain/edible oil/peanut/corn class feeds (premix), according to test paper
Bar shows that result determines positive cutoff value, shows this test strips positive cutoff value：≥5ng/ml.
2nd, sample preci-sion and accuracy is tested
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) it is used as precision evaluation index.Calculation formula is：The rate of recovery (%)=actual measured value/theoretical value × 100%, its
The addition concentration of middle theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, and X is
The average value of determination data.
By five concentration aflatoxin B1s of 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml respectively to paddy
The samples such as thing grain/edible oil/peanut/corn class feed (premix) are added recovery measure, and each sample does 4 and put down
OK, it is measured with three batches of different reagents, the average recovery rate and precision result for calculating sample see the table below.
The sample precision of table 1 and accuracy test
It is right respectively with the aflatoxin B1 of five concentration of 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml
The samples such as grain/edible oil/peanut/corn class feed (premix) are added, average recovery rate 91.8%~
Between 108.0%；Batch in, batch between relative standard deviation be respectively less than 15%.
3rd, cross reacting rate is tested
Selection with aflatoxin B1 there is similar structures and the medicine of similar functions to carry out time-resolved fluorescence test strips
Measure, its 50% inhibition concentration is respectively obtained by the standard curve of various medicines, and kit is calculated to other medicines with following formula
Cross reacting rate.
The cross reacting rate of table 2 is tested
| Medicine name | Cross reacting rate (%) |
| Aflatoxin B1 | 100 |
| Aflatoxin B 2 | 7.5 |
| Aflatoxins M1 | 2.2 |
| Aflatoxin M 2 | 4.3 |
| Aflatoxin G 1 | ＜ 1 |
| Aflatoxin G 2 | ＜ 1 |
Claims (7)
1. a kind of time-resolved fluorescence test strips for detecting aflatoxin B1, including sample absorption pad, reaction film and water suction
Pad, it is characterised in that the reaction film is overlapped in reaction film left and right ends respectively positioned at centre, sample absorption pad with adsorptive pads；Institute
State sample absorption pad and be provided with sample application zone and microballoon area, it is micro- that microballoon area is loaded with aflatoxin B1 time time-resolved fluorescence
Ball；The reaction film is provided with detection line (T lines) and nature controlling line (C lines), and T lines connect coating aflatoxin B1 antigen (coating
It is former), C lines coating anti-rabbit antibody, the aflatoxin B1 antigen is aflatoxin B1 hapten-carrier protein conjugate,
The preparation method of the aflatoxin B1 haptens is as follows：
31.2mg aflatoxin B1s are dissolved in 2ml ethanol, add 12mg sodium cyanoborohydrides, 0 DEG C of stirring and dissolving.Add
16.5mg 4- hydrazino-benzoic acids, stirring, 50 DEG C are to slowly warm up to, stir 1h.Solvent evaporated, column chromatography purifying, obtain 39mg whites
Solid, yield 88%.
2. the time-resolved fluorescence test strips of detection aflatoxin B1 as claimed in claim 1, it is characterised in that the Huang
Aspertoxin B1 haptens is to react to obtain by aflatoxin B1 and 4- hydrazino-benzoic acids, and its molecular structural formula is：
3. the time-resolved fluorescence test strips of detection aflatoxin B1 as claimed in claim 1, it is characterised in that the Huang
Aspertoxin B1 time-resolved fluorescence microballoons, including detection microballoon and Quality Control microballoon, detection microballoon are that pan coating has aspergillus flavus
The fluorescent microsphere of toxin B1 monoclonal antibodies, Quality Control microballoon are the fluorescent microsphere that pan coating has rabbit-anti labelled protein.
4. the time-resolved fluorescence test strips of detection aflatoxin B1 as claimed in claim 1, aflatoxin B1 Dan Ke
Grand antibody is prepared using aflatoxin B1 hapten-carrier protein conjugate as immunogene, the system of the immunogene
Preparation Method is as follows：
133mg BSA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Aflatoxin B1 haptens and BSA ratios can be 1: 1
Adjusted in the range of~60: 1, the application selection add 44.8mg aflatoxin B1s haptens (be 50 with BSA mol ratios:
1) EDC hydrochloride 10mg, are added, 2h is stirred at room temperature.The 48h that dialyses is moved into bag filter.
5. the time-resolved fluorescence test strips of detection aflatoxin B1 as claimed in claim 1, the preparation of the coating antigen
Method is as follows：
86mg OVA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Aflatoxin B1 haptens and OVA ratios can 1: 1~
Being adjusted in the range of 40: 1, the application selection is adding 35.8mg aflatoxin B1s haptens (being 40: 1 with OVA mol ratios),
EDC hydrochloride 10mg are added, 2h is stirred at room temperature.The 48h that dialyses is moved into bag filter.
6. a kind of method for preparing any one of the claim 1-5 test strips, it includes step：
1) the sample absorption pad for being coated with aflatoxin B1 monoclonal antibody-time-resolved fluorescence label is prepared；
2) prepare with the detection line for being coated with aflatoxin B1 hapten-carrier protein conjugate and be coated with anti-rabbit antibody
Nature controlling line reaction film；
3) test strips 1) will be assembled into sample absorption pad, reaction film and the adsorptive pads 2) prepared.
7. aflatoxin B1 remains in the samples such as one kind detection grain, edible oil, peanut, corn class feed (premix)
Method, it includes step：
1) Sample pretreatment；
2) detected with the test strips described in claim any one of 1-6；
3) testing result is analyzed.
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