CN106093381A - Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip - Google Patents
Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip Download PDFInfo
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Abstract
The present invention relates to food safety monitoring field, specifically disclose the immuno-chromatographic test paper strip of a kind of detection rod method toxin rod method phenol monomethyl ether (AME), including sample pad, gold conjugation pad, NC film, adsorptive pads and PVC base plate, gold colloidal AME monoclonal antibody complex it is coated with on gold conjugation pad, detection line and nature controlling line it is provided with on NC film, it is fixed with, on detection line, the AME OVA coupled antigen that concentration is 1mg/mL, nature controlling line is fixed with the sheep anti mouse two that concentration is 1mg/mL and resists.The present invention is to use the colloidal gold strip of rod method toxin A ME content in a step indirect competition immuno-chromatographic test paper strip technology fast qualitative or half-quantitative detection fruit and vegerable sample, detection sensitivity reaches 10ng/mL, and it is strong to detect specificity, simple to operate, economical and practical, it is suitable for Site Detection, can be widely applied to analysis detection and the rapid screening of AME in fruit and vegerable high-volume sample.
Description
Technical field
The invention belongs to field of food safety relates to immunity colloidal gold test paper strip and the field of mycotoxin residue detection.
Specifically, the present invention relates to one rod method toxin rod method phenol monomethyl ether (AME) residual be applicable to detection fruit and vegerable
Colloidal gold immuno-chromatography test paper strip.
Background technology
Rod method mycete belongs to filamentous fungi, is a kind of pathogen being prevalent in the food such as water fruits and vegetables and corruption
Raw bacterium, it can in the environment of low temperature and moisture growth and breeding, be therefore cause cold preservation or long-distance transport during water fruits and vegetables
Etc. the major microorganisms that food decay is rotten.Rod method phenol monomethyl ether (AME) is the main secondary metabolite of rod method mycete
One of, owing to it has mutagenicity, genotoxicity, carcinogenecity, it is possible to induction single stranded DNA and the fracture of double-stranded DNA, and also quilt
Being accredited as the strong inhibitor of Topoisomerase I and II, health can be caused by the food that picked-up rod method toxin infects
Serious harm.Therefore, AME becomes at present both at home and abroad scientist and studies one of more main rod method toxin.
But up to the present, in the food of domestic and foreign current, the limit standard of mycotoxin does not the most include rod method toxin,
But in recent years, the health risk of people and animals has been issued science suggestion with regard to food with rod method toxin in feedstuff by European Union, and
And in international trade, businessman entrusts third party to detect the phenomenon of rod method toxin the most generally, its detection is main to be used
The traditional detection such as gas chromatogram (GC) in laboratory, gas chromatography mass spectrometry (GC-MS), liquid chromatograph (LC), LC-MS (LC-MS)
Method.But, these traditional detection methods typically require large-scale, expensive instrument and equipment and technical professional, and
And detection can only be carried out in the lab, the detection time is long, wastes time and energy, it is impossible to meet what present food security field was pursued
In real time, quickly, the demand of portable inspectiont.
For the deficiency of rod method toxin A ME traditional detection method, we devise one and can be used for detecting water fruits and vegetables
Deng the colloidal gold immune chromatography test method of rod method toxin A ME in food, the method has quick, simple to operate, high special
Property, high sensitivity, testing cost are low, need not detecting instrument, and be particularly well-suited to on-the-spot great amount of samples real-time, quickly take out
Sample detects, it is possible to preferably auxiliary China's food enterprise and government function supervision department etc. carries out relevant detection work.
Summary of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of employing one-step method the most competing
Strive immuno-chromatographic test paper strip technology and quickly detect the colloidal gold strip of rod method toxin A ME residual, rod method in fruit and vegerable sample
The detection sensitivity of toxin A ME is up to 10ng/mL.
In order to realize the goal of the invention of the present invention, the invention provides a kind of detection rod method toxin rod method phenol monomethyl ether
(AME) immuno-chromatographic test paper strip, including sample pad, gold conjugation pad, NC film, adsorptive pads and PVC base plate, described NC film glues
Being attached on PVC base plate, gold conjugation pad and adsorptive pads stick to the two ends of NC film respectively, and sample pad sticks to gold colloidal and combines
The other end of pad；Gold colloidal-AME monoclonal antibody complex it is coated with on gold conjugation pad.
Further, the preparation method of described gold colloidal-AME monoclonal antibody complex is as follows: reduced by gold chloride with reducing agent
Become Au colloidal nanoparticles；Being mixed with AME monoclonal antibody by gold colloidal and hatch 30min, the BSA solution adding 10% makes it eventually
Concentration is 1%, reaction 20min after, add 10% PEG20000 and make its final concentration of 0.2%, continue reaction 20min；From
Heart purification, concentration produce gold colloidal-AME monoclonal antibody complex.Described reducing agent optimization citric acid trisodium.
Further, the preparation method of described AME monoclonal antibody is as follows:
(1) prepared by AME carboxylated derivative hapten: by the AME of 10mg, 14.5mg bromo butyric acid methyl ester and the carbon of 30.2mg
Acid potassium is dissolved in Methanamide (DMF) solution of 1mL, 50 DEG C of magnetic agitation reaction 2h；After the cooling of question response mixture, add 2mL water
Dilution, carries out precipitation intermediate product (15mg) is obtained by extraction with ethanol subsequently；Precipitation is dissolved in 0.5mL methanol and 0.5mL tetrahydrochysene
Furan (THF) mixed liquor, is subsequently added in the aqueous solution (0.1mL) containing 1.8mg Lithium hydrate, reacts under room temperature condition
5h, obtains the AME hapten (40mg) of carboxylated modification；
(2) prepared by AME-BSA antigen: 15.8mg AME hapten is dissolved in 1.5mL N,N-dimethylformamide (DMF)
In；Add 13.7mg dicyclohexylcarbodiimide (DCC), stir 30min；Add 7.6mg N-hydroxy-succinamide (NHS),
Room temperature lucifuge magnetic agitation 3.5h；Centrifugal, disgorging N, N '-dicyclohexylurea；It is added dropwise to supernatant be delayed by phosphoric acid
Rush in the BSA solution that liquid (0.1M, pH 7.4) is prepared, room temperature lucifuge stirring 2h；Dialyse 3d by the PBS solution of 0.1mol/L, often
It changes liquid 3 times, obtains AME-BSA antigen；After subpackage, it is stored in-20 DEG C of refrigerators standby；
(3) antibody screening and purification:
(a) animal immune: utilize AME-BSA antigen that the Balb/c mice of 8-10 week old carries out four minor tick immunity,
Carry out a booster immunization after 4th immunity, to swash intravital lymphocyte, produce AME high-affinity monoclonal anti
Body；B () cell merges and clone: will produce splenocyte and the myeloma of the Balb/c mice of AME high-affinity monoclonal antibody
Cell SP2/0 carries out cell fusion according to the ratio of 8:1, and utilizes indirect competitive ELISA method to measure cell supernatant, screening
Positive hole；Utilize limiting dilution assay to carry out sub-clone in the positive cell obtained after sensing 2 days, obtain stably dividing
Secrete the hybridoma of AME monoclonal antibody；The ascites preparation of (c) monoclonal antibody and purification: utilize liquid paraffin sensitization
Balb/c mice, and the hybridoma cell strain that step (2) obtains is carried out by 1640 culture medium, and it is injected in mice abdomen
Chamber；The ascites that will collect, is purified by octanoic acid-saturated ammonium sulfate method.
As preferably, the nano-particle size of described gold colloidal is 13～40nm, more preferably 18nm.
As preferably, described AME monoclonal antibody is 7.2 μ g/mL with the best combination amount of gold colloidal.
As preferably, described AME monoclonal antibody is 8.0 with the best combination pH value of gold colloidal.
Further, described NC film is provided with detection line and nature controlling line；The spacing of detection line and nature controlling line is 5mm.Its
Described in NC film preferred Millipore135 film.
Wherein, described detection line being fixed with AME-OVA coupled antigen, concentration is 1mg/mL.Fix on described nature controlling line
Having sheep anti mouse two to resist, concentration is 1mg/mL.
The coupling method of described AME-OVA coupled antigen can use this area routine coupling method.
As preferably, the present invention provides a preferably coating antigen synthetic method, and detailed process is as follows:
A () weighs aforementioned AME hapten 9.2mg, EDC 7.4mg, join in 2mL DMF solution, is allowed to fully dissolve,
Room temperature magnetic agitation reaction 20min, referred to as A liquid；
B () weighs oralbumin (OVA) 60mg and is dissolved in 7mL PBS, referred to as B liquid.
C A liquid is dropwise added drop-wise in B liquid by (), under the conditions of sealing lucifuge 4 DEG C, and magnetic stirrer over night.
D above-mentioned reactant liquor is transferred in pretreated bag filter by (), in 4 DEG C of chromatography cabinets, delay with 0.05mol/L PBS
Rushing liquid dialysis 3d, wherein, the every 8h of front 24h changes liquid 1 time.OVA-AME coating antigen is i.e. obtained after having dialysed.Product after dialysis is entered
Row subpackage, is positioned in-20 DEG C of refrigerators and saves backup.
It should be noted that those skilled in the art are on the basis of above-mentioned synthetic method, each material consumption is carried out
Ratio increases or reduces, all within protection scope of the present invention.
Described gold conjugation pad is glass fibre element film, and the nano-particle size of described gold colloidal is 13～40nm, excellent
Select 18nm.
Described sample pad is preferably glass fibre element film.
Described adsorptive pads is preferably filter paper fibre.
Further, the width of described test strips is 4～5mm.
As preferably, the other end of one end 2mm, the NC film that described adsorptive pads pushes down NC film is coated has gold colloidal-AME to resist
The gold conjugation pad of nanocrystal composition pushes down 2mm, and sample pad pushes down gold conjugation pad 2mm.
Further, the concrete preparation process of described colloidal gold immuno-chromatography test paper strip is:
(1) sample pad pretreatment: sample pad is positioned over pre-treatment buffer, and (0.5g BSA, 50 μ L Tween-20 are dissolved in
The phosphate buffer of 50mL) in, and hatch 30min in 37 DEG C of constant water bath box；Use substantial amounts of phosphate buffer subsequently
Thoroughly clean, 37 DEG C of baking ovens are dried 2h, save backup under last 4 DEG C of drying conditions.
(2) colloidal gold pad pretreatment: gold conjugation pad is immersed in pre-treatment buffer (1g BSA, 1.5g sucrose and
0.01g sodium azide is dissolved in the phosphate buffer of 50mL 10mM pH 7.4) in, and be positioned in 37 DEG C of constant water bath box and hatch
30min；Thoroughly clean with substantial amounts of phosphate buffer subsequently, 37 DEG C of oven drying 2h, protect under last 4 DEG C of drying conditions
Deposit standby；
(3) Au colloidal nanoparticles of aforementioned AME monoclonal antibody with 18nm is hatched formation gold colloidal-antibody to be combined
Thing, and be sprayed at pretreated gold conjugation pad, wherein every 1cm gold conjugation pad spraying 0.01mL gold colloidal-
AME antibody coupling complex solution；
(4) prepared by detection line T line: according to the package amount of 1.0 μ L/cm, AME-OVA coating antigen (1mg/mL) is fixed on NC
Film T line predeterminable area, forms T line；
(5) prepared by nature controlling line C line: equally according to the package amount of 1.0 μ L/cm, sheep anti mouse two anti-(1mg/mL) is fixed on NC
Film C line predeterminable area, forms C line；
(6) test strips assembles: first, be pasted onto on PVC base plate by the NC film being fixed with antibody；Secondly, adsorptive pads pressure is used
Live the coated gold conjugation pad pressure having gold colloidal-AME antibody complex of the other end of one end about 2mm, the NC film of NC film
Live in about about 2mm；Finally, sample pad pushes down gold conjugation pad about 2mm.
The invention provides a kind of method utilizing above-mentioned colloidal gold strip detection rod method toxin A ME, its detection is
The sample solution to be checked using Dispette to draw 70 μ about L drips on sample absorption pad, observes detection line and nature controlling line
Colour developing result.
The beneficial effects of the present invention is:
The present invention is from design AME hapten structure, preparation immunogen and coating antigen, and then screens high specificity, height
The AME monoclonal antibody of affinity, and be applied to further in the preparation of colloidal gold immuno-chromatography test paper strip.
The colloidal gold immuno-chromatography test paper strip of rod method toxin A ME in the detection fruit and vegerable of the present invention a, it is possible to step realizes inspection
Surveying, result is accurate, it is not necessary to washing and standard reference, it is possible to single or batch, sample is the most qualitative or half-quantitative detection,
Detection sensitivity is high, and detection limit reaches 10ng/mL.Molecular structure analog rod method phenol AOH is rung by described test strips without intersecting
Should.
Rod method toxin A ME colloidal gold immuno-chromatography test paper strip in the detection fruit and vegerable of the present invention, has preparation simple, price
Cheaply, it is possible to reduce because operating the lengthy and tedious detection error caused；It addition, the reagent holding time is long, execute-in-place is simple and convenient, can
At the scene high-volume sample plays a significant role in quickly detection and the examination of rod method toxin A ME.Can preferably assist
China's food enterprise and government function supervision department etc. carry out relevant detection work.
Accompanying drawing explanation
Fig. 1 is AME hapten synthesis route map of the present invention.
Fig. 2 is AME hapten hydrogen nuclear magnetic resonance spectrogram of the present invention.
Fig. 3 is AME hapten LC-MS spectrogram of the present invention.
Fig. 4 is AME colloidal gold immuno-chromatography test paper strip structural representation of the present invention.
Fig. 5 is AME colloidal gold strip testing result process decision chart of the present invention.
Fig. 6 is the colloidal gold immuno-chromatography test paper strip of the present invention testing result figure to AME.
Fig. 7 is the specific detection result figure of colloidal gold immuno-chromatography test paper strip of the present invention.
Detailed description of the invention
Below in conjunction with embodiment, the preferred embodiment of the present invention is described in detail.It will be appreciated that following reality
Execute providing merely to play descriptive purpose of example, be not used to the scope of the present invention is limited.The skill of this area
Art personnel, in the case of without departing substantially from spirit of the invention and spirit, can carry out various amendment and replacement to the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The preparation of embodiment 1AME monoclonal antibody
1, AME hapten synthesis
The potassium carbonate of the AME of 10mg, 14.5mg bromo butyric acid methyl ester and 30.2mg is dissolved in Methanamide (DMF) solution of 1mL,
50 DEG C of magnetic agitation reaction 2h；After the cooling of question response mixture, adding the dilution of 2mL water, it is heavy to carry out being obtained by extraction with ethanol subsequently
Shallow lake intermediate product (15mg)；Precipitation is dissolved in 0.5mL methanol and 0.5mL oxolane (THF) mixed liquor, be subsequently added to containing
In the aqueous solution (0.1mL) of 1.8mg Lithium hydrate, react 5h under room temperature condition, obtain the AME hapten of carboxylated modification
(40mg).The carboxylated modification of AME and nuclear magnetic resonance, NMR, LC-MS characterize spectrogram and see Fig. 1,2,3 respectively.
2, immunogenic synthesis
Immunogen of the present invention is that AME hapten obtains with BSA coupling.Concrete preparation method: by 15.8mg AME half
Antigen is dissolved in 1.5mL N,N-dimethylformamide (DMF)；Add 13.7mg dicyclohexylcarbodiimide (DCC), stirring
30min；Add 7.6mg N-hydroxy-succinamide (NHS), room temperature lucifuge magnetic agitation 3.5h；Centrifugal, disgorging N,
N '-dicyclohexylurea；Being added dropwise to by supernatant in the BSA solution prepared by phosphate buffer (0.1M, pH 7.4), room temperature is kept away
Light stirring 2h；Dialysis.With the PBS solution dialysis 3d of 0.1mol/L, change liquid every day 3 times, obtain AME-BSA immunogen, after subpackage,
It is stored in-20 DEG C of refrigerators standby.
3, the preparation of AME monoclonal antibody
(1) animal immune
The AME-BSA immunogen of synthesis is dissolved in PBS (pH 7.0), then by immunogen and Freund adjuvant according to
The ratio of 1:3 is fully emulsified makes oil emulsion vaccine.Immunity for the first time uses Freund's complete adjuvant vaccine, to 8-10 week old
Balb/c mice carries out immunity, carries out multi-point injection at neck dorsal sc, and inoculation dosage is 200 μ g time/only.15 days laggard
Row second time immunity, uses incomplete Freund's adjuvant, and immunizing dose is ibid；Carry out third time immunity after 30 days, use Freund not
Freund's complete adjuvant, immunizing dose is ibid；Carrying out the 4th immunity after 44 days, use incomplete Freund's adjuvant equally, immunizing dose is same
On；Carry out booster immunization after 58 days, be not added with adjuvant, directly use AME-BSA to carry out immunity.The time of immunity Balb/c mice sees
Table 1.
Table 1 immunity Balb/c mice timetable
(2) cell merges and clone
By can stably produce the splenocyte of the Balb/c mice of AME monoclonal antibody and myeloma cell SP2/0 according to
The ratio of 8:1 mixes, and is centrifuged subsequently, discards supernatant, and the cell of precipitation is made pasty state, is positioned over 37 DEG C of water baths, and
In 1min, add fusion agent Polyethylene Glycol (PEG4000), be gently mixed cell effect 2min, and add in 4min subsequently
The PEG nutritional solution of 20mL serum-free, 1000rpm discards supernatant after being centrifuged 10min.Resuspended carefully with 20mL-50mL complete culture solution
Born of the same parents, and it is inoculated in the 96 porocyte culture plates containing feeder cells, every hole 100 μ L, and it is placed on 37 DEG C of constant incubators
In；Bottom cell length to 96 porocyte culture plates 1/2～1/3 time, utilize indirect competitive ELISA method measure cell conditioned medium
Liquid, the positive hole of screening；Utilize limiting dilution assay, in positive cell 2d after sensing, carry out sub-clone, until obtain can
The hybridoma cell strain of stably excreting AME monoclonal antibody；It is enlarged this hybridoma cell strain cultivating and passing on, endless
Generation AME monoclonal antibody specific.
(3) preparation of monoclonal antibody ascites and purification
(1) mouse sensitization: utilize liquid paraffin sensitization Balb/c mice, after 6-8 week, the former 500 μ L/ of injecting immune, 10
Ascites can be prepared after it.
(2) injection cell: collect hybridoma and clean cell twice by 1640 culture medium, taking 100-150 ten thousand cell
Being injected in mouse peritoneal, after one week, visible mice state is inactive and the abdominal cavity enlargement of mice.
(3) ascites collection: injection cell, after a week, gathers ascites with asepsis injector to the mice of abdominal cavity enlargement, every
1d 2d gathers once, the most repeatedly gathers until mice natural death.
(4) ascites purification: by octanoic acid-saturated ammonium sulfate method, the ascites collected is purified, puts into-20 DEG C after purification
Refrigerator preserves.Before using, 0.01mol/L NaCl dialysis 48h need to be utilized under the conditions of 4 DEG C, change liquid once every 6h-8h.
4, the mensuration of antibody titer
In each hole of ELISA Plate, it is coated 100 μ L is respectively diluted to certain density coating antigen, hatch 2h for 37 DEG C；Clean
After liquid washes plate 3 times, every hole adds confining liquid 100 μ L, hatches 2h for 37 DEG C；After plate 3 times washed by cleanout fluid, every hole is separately added into by routine
The ELIAS secondary antibody that ELIAS secondary antibody diluted is 2000 times；Add the antibody 100 μ L of gradient dilution, 25 DEG C of lucifuge reactions
30min；After washing plate 4-5 time, add 50 μ L substrate solution A liquid, 50 μ L substrate solution B liquid；50 μ L stop buffers are added eventually after colour developing 30min
Only reaction；Measuring absorbance by microplate reader, absorbing wavelength is 450/630nm.The titer of the monoclonal antibody that the present invention obtains can
Reach 1:270000.
The preparation of embodiment 2AME colloidal gold immuno-chromatography test paper strip
The preparation of 2.1 gold colloidals
Take three aqueous solution of chloraurate of 99mL ultra-pure water and 1mL 1%, after mixing, magnetic agitation heated and boiled, add
1.8mL 1% citric acid three sodium solution, continues heated and boiled.When the color from pale yellow complexion changed of solution is black-and-blue, eventually become
After claret, continue heated and boiled 10min.When the temperature of the colloidal gold solution prepared is down to room temperature, the glue that will prepare
Body gold solution constant volume is to 100mL.Finally, the colloidal gold solution prepared is put in clean vial, and in 4 DEG C of environment bars
Deposit standby under part.
The preparation of 2.2 gold colloidal-AME antibody complexes
Take the colloidal gold solution 2mL that size is 18nm prepared, use 0.1M K2CO3Regulate its pH to 8.0；Subsequently
Being added dropwise over the AME monoclonal antibody (embodiment 1 gained) that 200 μ L concentration are 0.1 μ g/ μ L, under room temperature condition, magnetic agitation is anti-
Answer 2h；It is added dropwise over the HSA solution of 10% so that it is final concentration of 0.5%, magnetic agitation reaction 30min under room temperature condition；Add
The PEG20000 solution of 10%, and make its final concentration of 0.2%, magnetic agitation 30min under room temperature condition；12000rpm is centrifuged
30min, discards supernatant, stays gold colloidal to precipitate；The resuspended gold colloidal of HSA solution of 1%, then 12000rpm is added in precipitation
Centrifugal, after repeating three times, with gold colloidal re-suspension liquid (0.025g PEG20000,2.5g sucrose, 0.5g HSA and 50 μ L
Tween-20 is dissolved in the Tris-HCl buffer of 50mL 10mM pH 8.0) resuspended it is precipitated to the 1/10 of original volume.
2.3 gold colloidal-AME antibody complex being coated on gold conjugation pad
Gold conjugation pad is immersed in pre-treatment buffer, and (1g BSA, 1.5g sucrose and 0.01g sodium azide are dissolved in
The phosphate buffer of 50mL10mM pH 7.4) in, and be positioned in 37 DEG C of constant water bath box and hatch 30min；Subsequently with a large amount of
Phosphate buffer thoroughly clean, 37 DEG C of oven drying 2h, save backup under last 4 DEG C of drying conditions；Take above-mentioned system
The 10 μ L gold colloidal-AME antibody complexes got ready are laid on gold conjugation pad (1cm length × 0.5cm width), after 37 DEG C of baking oven 1h, 4
Save backup under DEG C drying condition.
The preparation of 2.4 sample pad
Sample pad is positioned over pre-treatment buffer, and (0.5g BSA, 50 μ L Tween-20 are dissolved in the phosphate-buffered of 50mL
Liquid) in, and hatch 30min in 37 DEG C of constant water bath box；Thoroughly clean with substantial amounts of phosphate buffer subsequently, 37 DEG C of baking ovens
In be dried 2h, save backup under last 4 DEG C of drying conditions.
2.5 nitrocellulose filters (NC film) upper detection line and the preparation of nature controlling line
Detection line T line: AME-OVA coating antigen (1mg/mL) is fixed on NC film T line according to the package amount of 1.0 μ L/cm pre-
If region, form T line；
Nature controlling line C line: equally sheep anti mouse two anti-(1mg/mL) is fixed on NC film C line according to the package amount of 1.0 μ L/cm pre-
If region, form C line；
Distance between nature controlling line and detection line is about 5mm, places 2h for 37 DEG C and is dried fixing.In order to prevent NC film
Non-specific adsorption, we are fixed with coating antigen and two anti-NC films are closed to dried: will be fixed with coating antigen with
Two anti-NC films are immersed in NC membrane closure liquid, and (1.2g HSA, 0.2g defatted milk powder and 120 μ L Tween-20 are dissolved in 40mL
Tris-HCl buffer) in, and it is put in 37 DEG C of calorstats maintenance 30min.With substantial amounts of PB buffer, it is carried out thorough subsequently
After the end is cleaned, it is dried under room temperature condition, deposits standby under last 4 DEG C of drying conditions.
The assembling of 2.6 test strips
First, the NC film being fixed with antibody is pasted onto on PVC base plate；Secondly, one end 2mm of NC film is pushed down with adsorptive pads
Left and right, the coated gold conjugation pad having gold colloidal-AME antibody complex of the other end of NC film pushes down about about 2mm；?
After, sample pad pushes down gold conjugation pad about 2mm.Colloidal gold immuno-chromatography test paper strip cross-sectional view is shown in Fig. 4.
Embodiment 3AME colloidal gold immuno-chromatography test paper strip Cleaning Principle
1, detection method: draw sample to be checked with Dispette, be added drop-wise to 3-4 and drip (about 70 μ L) in sample pad.
2, result judges:
Colloidal gold strip testing result judges to see Fig. 5.
Positive: T line does not develops the color, C line develops the color, and represents that in sample, the concentration of AME is higher than detection limit, and testing result is positive；
Negative: T line and C line all develop the color, represent that in sample, the concentration of AME is less than detection limit, testing result is negative；
Invalid: C line does not develops the color, represent the deterioration failure of this test strips, testing result is invalid.
The detection to AME of embodiment 4 colloidal gold immuno-chromatography test paper strip of the present invention
With AME standard substance for detection object, dilute AME standard substance successively with PB buffer so that it is concentration is respectively 50ng/
mL、40ng/mL、30ng/mL、20ng/mL、10ng/mL、5ng/mL、1ng/mL、0.1ng/mL、0ng/mL.Take respectively on 70 μ L
State AME standard substance, be added drop-wise to respectively on the colloidal gold immuno-chromatography test paper strip of preparation detect, after 5-10min, observe colour developing
Result (Fig. 6).
As can be seen from the results, when AME concentration is less than 10ng/mL, along with the reduction of AME standard concentration, detection
On line, red color intensity increases；When AME concentration is more than 10ng/mL, detection line does not develops the color.
The above results shows, the detection of this test strips is limited to 10ng/mL.
The specific test of embodiment 5 colloidal gold strip of the present invention
Owing to rod method phenol AOH with AME has similar structure, therefore, the present invention utilizes AOH to investigate this colloid further
The detection specificity of gold test paper strip.With AOH standard substance for detection object, dilute AOH standard substance successively with PB buffer so that it is dense
Degree 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 0ng/mL respectively.Take 70 μ L above-mentioned AOH standard solution successively,
It is added drop-wise to respectively on the colloidal gold immuno-chromatography test paper strip of preparation detect, after 5-10min, observes colour developing result (Fig. 7).
Result is shown as: when the concentration of AOH is respectively 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 0ng/mL
Time, detecting line and nature controlling line all develops the color, testing result is feminine gender, it can be deduced that the colloidal gold immuno-chromatography test paper strip of the present invention
There is high detection specificity, no cross reaction.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. the immuno-chromatographic test paper strip of detection rod method toxin rod method phenol monomethyl ether (AME), combines including sample pad, gold colloidal
Pad, NC film, adsorptive pads and PVC base plate, it is characterised in that on gold conjugation pad, be coated with gold colloidal-AME monoclonal antibody complex.
Colloidal gold strip the most according to claim 1, it is characterised in that be provided with detection line and nature controlling line on NC film；
Being fixed with AME-OVA coupled antigen on described detection line, concentration is 1mg/mL.
Colloidal gold strip the most according to claim 2, it is characterised in that be fixed with sheep anti mouse two on described nature controlling line and resist,
Concentration is 1mg/mL.
4. according to colloidal gold strip described in any one of claims 1 to 3, it is characterised in that described gold colloidal-AME monoclonal antibody is multiple
The preparation method of compound is as follows: with reducing agent, gold chloride is reduced into Au colloidal nanoparticles；By gold colloidal and AME monoclonal anti
30min is hatched in body mixing, add the BSA solution of 10% make its final concentration of 1%, after reaction 20min, add 10%
PEG20000 and make its final concentration of 0.2%, continue reaction 20min；Centrifugal purification, concentration produce gold colloidal-AME monoclonal antibody and are combined
Thing.
Colloidal gold strip the most according to claim 4, it is characterised in that the preparation method of described AME monoclonal antibody is such as
Under:
(1) prepared by AME carboxylated derivative hapten: AME, bromo butyric acid methyl ester and potassium carbonate are dissolved in formamide solution, 50 DEG C
Magnetic agitation reaction 2h；After the cooling of question response mixture, dilute, carry out being obtained by extraction in the middle of precipitation with ethanol subsequently and produce
Thing；Precipitation is dissolved in methanol and oxolane mixed liquor, is subsequently added in the aqueous solution containing Lithium hydrate, under room temperature condition
Reaction 5h, obtains the AME hapten of carboxylated modification；
(2) prepared by AME-BSA antigen: be dissolved in N,N-dimethylformamide by AME hapten；Add dicyclohexylcarbodiimide
Stirring；Add N-hydroxy-succinamide, room temperature lucifuge magnetic agitation；Centrifugal, disgorging N, N '-dicyclohexylurea；Will
Supernatant is added dropwise in the BSA solution prepared by phosphate buffer, and room temperature lucifuge stirs；Saturating by the PBS solution of 0.1mol/L
Analysis 3d, changes liquid every day 3 times, obtains AME-BSA antigen；
(3) antibody screening and purification.
Colloidal gold immuno-chromatography test paper strip the most according to claim 4, it is characterised in that described AME monoclonal antibody with
The best combination amount of gold colloidal is 7.2 μ g/mL.
Colloidal gold immuno-chromatography test paper strip the most according to claim 4, it is characterised in that described AME monoclonal antibody with
The best combination pH value of gold colloidal is 8.0.
8. according to colloidal gold immuno-chromatography test paper strip described in claim 6 or 7, it is characterised in that the nanometer of described gold colloidal
Grain size is 13～40nm.
Colloidal gold immuno-chromatography test paper strip the most according to claim 8, it is characterised in that the nano-particle of described gold colloidal is big
Little for 18nm.
10. according to the colloidal gold immuno-chromatography test paper strip described in claims 1 to 3 or 5～7 any one, it is characterised in that described
The width of test strips is 4～5mm.
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| CN108675981A (en) * | 2018-05-04 | 2018-10-19 | 四川国际旅行卫生保健中心 | The virtual template molecular compound of the mould toxin of rod method and the molecularly imprinted polymer prepared using the compound |
| CN108776219A (en) * | 2018-02-12 | 2018-11-09 | 北京农业质量标准与检测技术研究中心 | A kind of immuno-chromatographic test paper strip of the thin Alternaria alternata ketone acid of quick detection |
| CN109799332A (en) * | 2018-12-25 | 2019-05-24 | 北京农业质量标准与检测技术研究中心 | The magnetic immunofluorescence quantitative detecting method of rod method phenol monomethyl ether |
| CN109900899A (en) * | 2019-04-08 | 2019-06-18 | 北京农业质量标准与检测技术研究中心 | Rod method phenol monomethyl ether micro-fluidic detection chip and detection method based on immunomagnetic isolation |
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| CN113109559A (en) * | 2021-03-23 | 2021-07-13 | 宁波市农业科学研究院 | Immunochromatography gold-labeled test strip suitable for trifloxystrobin pesticide and rapid test method thereof |
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| CN109900899A (en) * | 2019-04-08 | 2019-06-18 | 北京农业质量标准与检测技术研究中心 | Rod method phenol monomethyl ether micro-fluidic detection chip and detection method based on immunomagnetic isolation |
| CN112098641A (en) * | 2020-08-17 | 2020-12-18 | 北京勤邦生物技术有限公司 | Application of alternan artificial antigen in enzyme linked immunosorbent assay kit |
| CN112098641B (en) * | 2020-08-17 | 2023-07-11 | 北京勤邦科技股份有限公司 | Application of inter-alternaria alternata artificial antigen in enzyme-linked immunosorbent assay kit |
| CN113109559A (en) * | 2021-03-23 | 2021-07-13 | 宁波市农业科学研究院 | Immunochromatography gold-labeled test strip suitable for trifloxystrobin pesticide and rapid test method thereof |
| CN113588955A (en) * | 2021-07-08 | 2021-11-02 | 上海市农产品质量安全中心 | Test paper for alternaria toxin |
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