CN103257230B - Immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, preparation method and application thereof - Google Patents
Immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, a preparation method and application thereof. The test strip includes a paperboard. An absorbent pad, a detection pad, a gold labeled pad and a sample pad are sticked on one side of the paperboard from top to bottom. Adjacent pads are in overlapping connection at joints. The detection pad adopts a nitrocellulose membrane as a base pad, which is provided with a transverse control line and detection lines. The two detection lines in internal distribution are positioned below the quality control line, and are respectively coated with a zearalenone-bovine serum albumin conjugate and an aflatoxin B1-bovine serum albumin conjugate. The quality control line is coated with a rabbit antimouse polyclonal antibody. The gold labeled pad is horizontally sprayed with a nanogold labeled anti-aflatoxin universal monoclonal antibody and a nanogold labeled anti-zearalenone monoclonal antibody. The immunochromatographic test strip can be used for simultaneously detecting the content of fungaltoxins aflatoxin and zearalenone in a sample, and has the characteristics of simple operation, rapidity, and high sensitivity.
Description
Technical field
The present invention relates to the mycotoxin immuno-chromatographic test paper strip, be specifically related to synchronously detect immuno-chromatographic test paper strip, preparation method and the application thereof of aflatoxin and zearalenone composite pollution.
Background technology
Aflatoxin and zearalenone are the objectionable impuritiess be present in cereal, are the secondary metabolites that the fungi by Polluted grains produces.Grain and feed are not too high by temperature or humidity in abundant drying or transporting procedures in the results process, all may provide suitable condition for the growth and breeding of fungi, thereby cause the pollution of mycotoxin.Aflatoxin can destroy the liver organization of humans and animals strongly, when serious, can cause liver cancer even dead; Zearalenone has the oestrogen-like hormone effect, can cause the animal acute and chronic poisoning, causes that Reproduction is extremely even dead, and these two kinds of toxin are prevalent in grain and feed, and the mankind's health and safety is caused to serious threat.In view of the damaging effect of these two kinds of mycotoxins and the extensive generation in grain and feed thereof, in countries in the world grain and feed, the content of these two kinds of mycotoxins has carried out strict restriction.In order to strengthen the detection to these mycotoxins in grain and feed, ensure people's consumption safety, exploitation is for detection technique, the especially fast detecting of these mycotoxins, be to understand and grasp food and feeds safety and sanitation information, strengthen the important step of food security consumption.
The existing detection method to aflatoxin and zearalenone mainly comprises thin layer chromatography, exact instrument analytic approach and immune analysis method.When thin layer chromatography detects mycotoxin, do not need special instrument and equipment, in common laboratory, all can carry out, but detect that reagent dosage is large, complex operation, other component serious interference, poor accuracy, can not accurate quantitative analysis, and larger to experimenter and surrounding environment contamination hazard, be unsuitable for field quick detection, its range of application is more and more narrow.The exact instrument analytic approach comprises the method for high performance liquid chromatography, liquid chromatography and mass spectrum and tandem mass spectrum coupling, it is highly sensitive, accuracy is good, but the instrument costliness, the degree of purification of the sample that requirement detects is high, sample pretreatment process is loaded down with trivial details, length consuming time, require highly to experimental situation and testing staff, be difficult to realize fast detecting, testing cost is high, is not suitable for field quick detection.Immune analysis method has overcome the shortcoming of thin layer chromatography and instrumental method, due to its high specificity, highly sensitive, sample pre-treatments is simple, cost is low, little to the contamination hazard of experimenter and surrounding environment, be suitable for the advantages such as on-the-spot batch detection and obtained fast development in recent years.Immunochromatography technique based on colloidal gold labeled monoclonal antibody and antigentic specificity association reaction is because its testing result naked eyes are visible, do not need large-scale instrument and equipment, testing cost is low, analysis time is short, on qualitative, online, the fast detecting of the minimal residue things such as mycotoxin, is widely applied in recent years.Yet the existing colloidal gold immuno-chromatography test paper strip detected for mycotoxin mostly can only detect a kind of mycotoxin.And the planting patterns of China smallholder decentralized, in agricultural product, the mycotoxin incidence is high, and the possibility that same agricultural product are polluted by multiple mycotoxin is large, therefore in the urgent need to synchronously detecting the detection technique of multiple mycotoxin, to realize synchronous, the fast monitored to mycotoxin composite pollution in grain and feed etc.
Summary of the invention
Problem to be solved by this invention is to provide immuno-chromatographic test paper strip, preparation method and the application thereof of a kind of synchronous detection aflatoxin and zearalenone composite pollution.This immuno-chromatographic test paper strip can be used for the synchronous detection of aflatoxin and two kinds of mycotoxin levels of zearalenone in sample, has simple to operate, quick, highly sensitive characteristics.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is:
The synchronous immuno-chromatographic test paper strip (seeing Fig. 1 and Fig. 2) that detects aflatoxin and zearalenone composite pollution, comprise cardboard, the one side of cardboard is pasted adsorptive pads from top to bottom successively, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, described detecting pad be take nitrocellulose filter as base wad, nitrocellulose filter is provided with horizontal nature controlling line and detection line, described detection line is positioned at the below of nature controlling line, number is two, be spaced apart, be coated with respectively zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) on described two detection lines, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, described gold mark pad transverse jet scribbles the anti-zearalenone monoclonal antibody of aspergillus flavus resisting toxin general purpose single clonal antibody and the nano gold mark of nano gold mark, the hybridoma cell strain 1C11 secretion that described aspergillus flavus resisting toxin general purpose single clonal antibody is CCTCC NO. C201013 by deposit number produces, and the hybridoma cell strain 2D3 secretion that anti-zearalenone monoclonal antibody is CCTCC NO. C201328 by deposit number produces, this hybridoma cell strain 2D3 has been preserved in Chinese Typical Representative culture collection center (CCTCC) on March 7th, 2013, the preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO. C201328.
Press such scheme, the long 16～18mm of described adsorptive pads, wide 3～4mm, the long 18～30mm of detecting pad, wide 3～4mm; Long 10～the 12mm of gold mark pad, wide 3～4mm; Long 12～the 15mm of sample pad, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm.
Press such scheme, described adsorptive pads is thieving paper.
Press such scheme, the spacing on described detecting pad between two detection lines is 2-4mm, near the spacing on edge on the detection line of nature controlling line and nitrocellulose filter, is 15～20mm, and the detection line of described close nature controlling line and the spacing of nature controlling line are 5～7mm.
Press such scheme, the package amount that described detecting pad is coated with every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) on the detection line of zearalenone-bovine serum albumin(BSA) conjugate is 100～300ng; On the detection line of coated aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA), the package amount of every centimetre of needed aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 100～300ng; On nature controlling line, the package amount of every centimetre of anti-mouse polyclonal antibody of needed rabbit is 50～200ng.
Press such scheme, in described gold mark pad, the particle diameter of nm of gold used is 15～20nm; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the upper every centimetre of spraying length of described gold mark pad is required is 100～200ng, and the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 200～400ng.
Detect simultaneously and rapidly as mentioned above the preparation method of the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution, comprise the following steps:
(1) preparation of adsorptive pads
Thieving paper is cut out and is obtained adsorptive pads;
(2) preparation of detecting pad
Being coated with of detection line:
The conjugate (AFB1-BSA) of zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and aflatoxin B1-bovine serum albumin(BSA) is mixed with respectively to the coating buffer of 0.25～0.5mg/mL with coated damping fluid, by a spray mode, it is coated with respectively on nitrocellulose filter, obtain two detection lines, then under 37～40 ℃ of conditions dry 30～60 minutes; On the detection line of described coated zearalenone-bovine serum albumin(BSA) conjugate, the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) is 100～300ng; On the detection line of coated aflatoxin B1-bovine serum albumin(BSA) conjugate, the package amount of needed aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 100～300ng, spacing between described two detection lines is 2-4mm, and on the detection line of close nature controlling line and nitrocellulose filter, the spacing on edge is 15～20mm;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with to the solution of 0.2～0.4mg/mL with coated damping fluid, position in the detection line 5～7mm apart near nature controlling line, by a spray mode, by it, laterally be coated on nitrocellulose filter, obtain nature controlling line, on every centimetre of nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50～200ng, then under 37～40 ℃ of conditions dry 1～2 hour;
(3) preparation of sample pad
Glass fibre membrane is put into to confining liquid and soak, take out, under 37～40 ℃ of conditions, drying is 6～10 hours, obtains sample pad, then puts room temperature preservation in exsiccator;
(4) preparation of gold mark pad
Glass fibre membrane is put into to confining liquid to soak, take out, under 37～40 ℃ of conditions, drying is 6～10 hours, by a spray mode to the horizontal mixed solution of the anti-zearalenone monoclonal antibody solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of spraying nano gold mark and nano gold mark on dry glass fibre membrane, wherein: every centimetre of consumption that sprays the aspergillus flavus resisting toxin general purpose single clonal antibody of the required nano gold mark of length is 100～200ng, the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 200～400ng, then vacuum freeze drying is 2～4 hours, put room temperature preservation in exsiccator, the hybridoma cell strain 1C11 secretion that described aspergillus flavus resisting toxin general purpose single clonal antibody is CCTCC NO.C201013 by deposit number produces, and the hybridoma cell strain 2D3 secretion that described anti-zearalenone monoclonal antibody is CCTCC NO.C201328 by deposit number produces,
(5) assembling of test strips
Paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad in the one side of cardboard, adjacent each pad overlapping connection in junction, overlapping length is 1～3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone composite pollution.
Press such scheme, contain in the every 10mL of described coated damping fluid: bovine serum albumin(BSA) 0.1～0.2g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g.
Press such scheme, contain in the every 100mL of confining liquid that described step (3) is used: bovine serum albumin(BSA) 1～2g, sucrose 2～3g, sodium azide 0.02～0.05g, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
Press such scheme, in the every 100mL of confining liquid that described step (4) is used, contain: bovine serum albumin(BSA) 1～2g, sucrose 2～3g, sodium chloride 1～2g, Tween-20 0.05～0.1g, polyvinylpyrrolidone 0.2～0.5g, sodium azide 0.02～0.05g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
Press such scheme, the general monoclonal antibody solution of the aspergillus flavus resisting toxin of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.4mL0.1mol/L wet chemical, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirred, continue to stir 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with the mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby;
The anti-zearalenone monoclonal antibody solution of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.425mL0.1mol/L wet chemical, the anti-zearalenone monoclonal antibody aqueous solution that slowly adds 2.5mL0.1mg/mL under the state stirred, continue to stir 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with the mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby;
Described 0.1mol/L wet chemical is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: the 2.0g PEG-400, and the 0.2g Sodium azide, 0.1235g boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained.
The application of the immuno-chromatographic test paper strip of synchronous detection aflatoxin as above and zearalenone composite pollution, method is as follows: take levigate testing sample, add the methanol aqueous solution that volumetric concentration is 60～80%, mix, under 50～60 ℃ of water-baths, ultrasonic extraction is 5～10 minutes, standing 5～10 minutes, by supernatant liquor, it is the extract dilute with water, the final volume concentration that makes methyl alcohol in dilution is 20～30%, obtain testing sample solution, get again this testing sample solution of 80-150 μ L as being detected on the sample pad of immuno-chromatographic test paper strip that detects liquid and dropwise join synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, separately get the consistent methanol aqueous solution of isopyknic methanol concentration as negative controls, dropwise add on the sample pad of another immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone composite pollution, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar: when on test strip, the coated zearalenone-detection line color of bovine serum albumin(BSA) conjugate (ZEA-BSA) approaches with the color of corresponding detection line on the control stripes bar, show in testing sample solution that zearalenone content is lower than 1ng/mL, during than corresponding detection line of light color, show in testing sample solution that zearalenone content is equal to or higher than 1ng/mL and lower than 4ng/mL, while not developing the color, show that in testing sample solution, the content of zearalenone is equal to or higher than 4ng/mL,
When the detection line color of the conjugate (AFB1-BSA) of coated aflatoxin B1-bovine serum albumin(BSA) approaches with the color of corresponding detection line on the control stripes bar on test strip, show in testing sample solution that aflatoxin content is lower than 0.25ng/mL; During than corresponding detection line of light color, show in testing sample solution that aflatoxin content is equal to or higher than 0.25ng/mL and lower than 1ng/mL; While not developing the color, show that in testing sample solution, the content of aflatoxin is equal to or higher than 1ng/mL;
When nature controlling line does not develop the color, no matter whether the detection line of test strip develops the color, and it is invalid that this test strips is judged to,
Finally by converting and obtaining the content of aflatoxin and zearalenone in testing sample.
This immuno-chromatographic test paper strip is in aflatoxin and zearalenone composite pollution are synchronous in detecting principle of work: when testing sample solution joins on the sample pad of test strips lower end, testing sample solution moves to the adsorptive pads direction along test strips by capillary action, when it moves to gold mark pad, the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark and the anti-zearalenone monoclonal antibody of nano gold mark are dissolved.While in sample, containing aflatoxin, aflatoxin will with gold mark pad on nano gold mark aspergillus flavus resisting toxin general purpose single clonal antibody in conjunction with and together upwards swimming, when its arrival is fixed wtih the detection line of aflatoxin B1-bovine serum albumin(BSA) conjugate antigen, antigen will be competed limited antigen binding site on the aspergillus flavus resisting toxin general purpose single clonal antibody of combining nano gold mark with aflatoxin, in sample, aflatoxin content is higher, antigen on detection line can in conjunction with the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark will be fewer, the colour developing band color formed on detection line is more shallow, while in sample, containing zearalenone, zearalenone will with gold mark pad on nano gold mark anti-zearalenone monoclonal antibody in conjunction with and together upwards swimming, when its arrival is fixed wtih the detection line I of zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) antigen, antigen will be competed limited antigen binding site on the anti-zearalenone monoclonal antibody of combining nano gold mark with zearalenone, in sample, zearalenone content is higher, antigen on detection line can in conjunction with the anti-zearalenone monoclonal antibody of nano gold mark will be fewer, the colour developing band color formed on detection line is more shallow.When the antibody of the correspondence of the nano gold mark of the antigen institute combination on two detection lines is less than certain quantity, two detection line places will not have red lines and occur.No matter in sample, whether contain this two kinds of mycotoxins, the antibody of the antibody of the antimycotic toxin of the nano gold mark that the antigen on not tested survey line is intercepted and captured or the antimycotic toxin of nano gold mark moves to nature controlling line with the bond of mycotoxin by continuing and the anti-mouse polyclonal antibody of the rabbit on nature controlling line is combined and is developed the color by enrichment.Accordingly, respectively the detection line of the conjugate of coated aflatoxin B1-bovine serum albumin(BSA) and the detection line of coated zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) on test strip are developed the color and contrast with corresponding detection line color on the control stripes bar, can obtain the composite pollution situation of aflatoxin and these two kinds of mycotoxins of zearalenone in sample.
Beneficial effect of the present invention:
(1) one step, detect aflatoxin and zearalenone simultaneously.Immuno-chromatographic test paper strip provided by the invention can be realized synchronous, the fast detecting to aflatoxin and two kinds of mycotoxins of zearalenone on a test strips, the antibody used is monoclonal antibody, specificity is good, highly sensitive, each mycotoxin is noiseless between detecting, simple, quick.
(2) highly sensitive.Immuno-chromatographic test paper strip provided by the invention is limited to 0.25ng/mL to the lowest detection that detects aflatoxin in solution, and the lowest detection of zearalenone is limited to 1ng/mL, and this detectability can meet the limit the quantity of requirement of European Union to these two kinds of mycotoxins in food.
(3) simple to operate.Sample pre-treatments only need to add the methanol-water extract in sample ultrasonic extraction 5～10 minutes, and then standing 5～10 minutes, to get the supernatant dilution and can be detected, whole sample pretreatment process is simple, fast.The accompanying drawing explanation
The front elevation of the immuno-chromatographic test paper strip that Fig. 1 is synchronous detection aflatoxin of the present invention and zearalenone composite pollution;
The side view of the immuno-chromatographic test paper strip that Fig. 2 is synchronous detection aflatoxin of the present invention and zearalenone composite pollution;
The process decision chart as a result that Fig. 3 is embodiment 2;
In figure: 1 cardboard, 2 adsorptive pads; 3 detecting pads; 4 gold medal mark pads; 5 sample pad; 6 nature controlling lines; 7 detection line I; 8 detection line II; 9 control stripes bars; 10 test strip.
Embodiment
Embodiment 1: the acquisition of aspergillus flavus resisting toxin general purpose single clonal antibody and anti-zearalenone monoclonal antibody
A. the hybridoma cell strain 1C11 secretion that aspergillus flavus resisting toxin general purpose single clonal antibody is CCTCC NO.C201018 by deposit number produces, the method of reporting in the patent that is specifically CN201010245095.5 according to number of patent application makes in advance, the preparation method is: the BALB/c mouse that hybridoma cell strain 1C11 injection was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt the caprylic acid-ammonium antibody purification, concrete operations are: with double-deck Filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, slowly add caprylic acid under stirring, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon precipitation, by the supernatant that obtains with after double-deck Filter paper filtering, the volumetric molar concentration that adds 1/10 filtrate volume is the phosphate buffer that 0.1mol/L and pH value are 7.4, regulate the pH value to 7.4 of this mixed liquor with the sodium hydroxide solution of 2mol/L, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon supernatant, the gained precipitation is resuspended with the 0.01mol/L phosphate buffer of former ascites volume 1/10, the bag filter of packing into, pure water is dialysed, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards the freeze drier freeze-drying, collect freeze-dried powder, obtain the aspergillus flavus resisting toxin general purpose single clonal antibody that purifying is good, antibody is placed in to-20 ℃ of refrigerators standby,
Described acetate buffer is the 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffer of described 0.1mol/L is 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g, add water and be settled to the 100mL gained.
B. the hybridoma cell strain 2D3 that anti-zearalenone monoclonal antibody is CCTCC NO.C201328 by deposit number produces, and its concrete preparation method is:
The BALB/c mouse that hybridoma cell strain 2D3 injection was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt the caprylic acid-ammonium antibody purification, concrete operation step is: with double-deck Filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, slowly add caprylic acid under stirring, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon precipitation, by the supernatant that obtains with after double-deck Filter paper filtering, the volumetric molar concentration that adds 1/10 filtrate volume is the phosphate buffer that 0.1mol/L and pH value are 7.4, regulate the pH value to 7.4 of this mixed liquor with the sodium hydroxide solution of 2mol/L, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon supernatant, 0.01mol/L by the gained precipitation with former ascites volume 1/10, the phosphate buffer that the pH value is 7.4 is resuspended, the bag filter of packing into, pure water is dialysed, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards the freeze drier freeze-drying, collect freeze-dried powder, obtain the anti-zearalenone monoclonal antibody that purifying is good, antibody is placed in to-20 ℃ of refrigerators standby,
Described acetate buffer is the 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffer of described 0.1mol/L is 8g sodium chloride, the 2.9g disodium hydrogen phosphate, and 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g, add the water constant volume to the 100mL gained; The phosphate buffer of described 0.01mol/L is 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g, add water and be settled to the 100mL gained.
The hypotype of identifying the anti-zearalenone monoclonal antibody of hybridoma cell strain 2D3 secretion with commercially available hypotype identification kit is IgG2b.
The BALB/c mouse ascites that records injection hybridoma cell strain 2D3 by the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method is purified the tiring of antibody obtained and can be reached 1.5 * 10
5, i.e. anti-zearalenone monoclonal antibody dilution 1.5 * 10
5times the time the measured in solution result positive.Identify that by conventional indirect competitive ELISA method its sensitivity to zearalenone is 20pg/mL, with β-zearalenol, α-zearalenol, the cross reaction of β-ZER is respectively 84.9%, 3.3%, and 3.2%.
Wherein: according to following methods, screening obtains above-mentioned hybridoma cell strain 2D3:
(1) animal immune
Buy 6 of BALB/c mouse in 6 week age, the zearalenone comlete antigen ZEA-BSA that immunity is commercially available.Immunity is by after zearalenone comlete antigen and the emulsification of isopyknic Fu Shi Freund's complete adjuvant, in the subcutaneous multi-point injection in mouse carotid back for the first time.Carry out after being immune to for the second time 4 weeks, adopt freund 's incomplete adjuvant and the emulsification of isopyknic zearalenone comlete antigen, inject in mouse peritoneal.Immunity for the third time and immune interval for the second time 4 weeks, immunization ways is identical with it, carries out after being immune to immune 3 weeks for the third time for the 4th time, and immunization ways, with immune identical for the second time, is similarly lumbar injection.4 times immunizing dose is identical, is every mouse 100 μ g.Latter 8 days of 3 times each immunity, tail vein blood, separation of serum, adopt indirect elisa method monitoring mice serum to tire.Latter 8 days of the 4th immunity, tail vein blood, separation of serum, adopt indirect elisa method monitoring mice serum to tire, and measure mice serum sensitivity by the indirect competitive ELISA method, selection is tired, sensitivity all relatively high mouse corresponding to serum carry out last booster immunization, immunizing dose is front 2 times.Zearalenone comlete antigen ZEA-BSA is purchased from German aokin company.
(2) Fusion of Cells
In last booster immunization after 3 days, adopting the polyglycol of 50% (percent by weight) is that PEG (molecular weight is 1450) makes fusion agent, carry out according to a conventional method Fusion of Cells, concrete steps: under aseptic condition, kill immune mouse, separating Morr. cell, with mouse source myeloma cell SP2/0 with 5～8: 1 number is than mixing, wash cell mixing with the RPMI-1640 basic culture solution, with 50%PEG, merge, merge 1 minute, then slowly add the RPMI-1640 basic culture solution, centrifugal, remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended containing the cell complete medium of 1%HAT with 20mL, the cell hanged is joined in the 80mL semisolid culturemedium, after mixing, be added on 6 porocyte culture plates, 1.5mL/ hole, being placed in 37 ℃ of CO2gas incubator cultivates.The described cell complete medium containing 1%HAT contains 20% (percent by volume) hyclone, 75% (percent by volume) RPMI-1640 basic culture solution, 1% (percent by weight) Glu, 1% (percent by volume) HEPES, 1% (percent by volume) two anti-(the 10000 every ml penicillins of unit and every milliliter of streptomysins of 10000 micrograms), 2% (percent by weight) growth factor (HFCS) and 1% (percent by weight) hypoxanthine-aminopterin-thymidine is HAT; Semisolid culturemedium is the cell complete medium containing 1% (mass percent) methylcellulose; RPMI-1640 basic culture solution, HEPES, two anti-and Glu are purchased from Hyclone company; 1% hypoxanthine-aminopterin-thymidine is that HAT and methylcellulose are purchased from Sigma-Aldrich company.
(3) screening of cell line and clone
2-3 week after Fusion of Cells, cell colony grows to people's naked eyes when visible, to clone from this nutrient culture media and draw with micropipettor, moving to 96 porocyte culture plates adopts liquid to amplify cultivation, every hole moves into 1 clone, at the bottom of cell grows to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, carry out antibody test.Adopt the ELISA method to be screened the culture hole that Growth of Hybridoma Cell is arranged, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out anti-zearalenone and the positive hole of not anti-carrier protein BSA; The positive hole that second step adopts the indirect competitive ELISA method to filter out the first step is detected, former as competition with zearalenone, all (the higher finger competition of light absorption value was that 0 hole is that the final tested volume in positive control hole is higher originally, competition original content that is IC when the higher finger inhibiting rate of sensitivity is 50% higher hole to select light absorption value and sensitivity
50be worth less), adopt limiting dilution assay to carry out subclone, after subclone, adopt same two-step approach to be detected, after so repeating subclone 2-3 time, acquisition hybridoma cell strain 2D3.
Hybridoma cell strain 2D3 antibody variable region sequencing
(1) extract total RNA: adopt the total RNA extraction reagent box of day root company and extract to specifications total RNA that can produce hybridoma cell strain 2D3;
(2) synthetic cDNA: total RNA that the step 1 of take obtains is template, oligo (dT)
15for primer, according to SuperScript
tM-2II reverse transcriptase instructions carries out reverse transcription, synthetic cDNA the first chain; Primer oligo (dT)
15by Invitrogen, buied;
(3) PCR method clone variable region gene: according to the conservative site design primer of GENEBANK small mouse antibody gene sequence, take cDNA as masterplate amplification antibody is light, heavy chain variable region gene.The PCR program is: 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min, and increase 30 and circulate, last 72 ℃ are extended 10min.The PCR product is after the agarose gel electrophoresis separation of 1% (percent by weight), purify and reclaim DNA fragmentation with kit, be connected in carrier pMD18-T, transform the bacillus coli DH 5 alpha competent cell, the picking positive colony, deliver to Shanghai Sani's bio tech ltd and checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is that (22mer) with 5 '-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3 ', (32mer) wherein S, M, R and W are the merger base to 5 '-AGG TSM ARC TGC AGS AGT CWG G-3 ', M=A/C, R=A/G, S=C/G, W=A/T, the variable region of light chain primer be 5 '-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3 ' (24mer) and 5 '-CCG TTT CAG CTC CAGCTT GGT CCC-3 ' (24mer).
The gene order result obtained: the long 315bp of variable region of heavy chain coding gene sequence, sequence is as shown in SEQ ID NO:1, derive the coded variable region of heavy chain of this gene order according to obtained gene order and be comprised of 105 amino acid, sequence is as shown in SEQ ID NO:3.The long 329bp of variable region of light chain coding gene sequence, sequence, as shown in SEQ ID NO:2, is derived the coded variable region of light chain of this gene order according to obtained gene order and is comprised of 109 amino acid, and sequence is as shown in SEQ IDNO:4.
The synchronous preparation method who detects the immuno-chromatographic test paper strip of aflatoxin and two kinds of mycotoxins of zearalenone, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out to growth 16mm, and the specification of wide 4mm, obtain adsorptive pads;
(2) preparation of detecting pad
Being coated with of detection line:
Zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) is mixed with to the coating buffer of 0.4mg/mL with coated damping fluid, on the distance nitrocellulose filter along the position of 15mm, by a spray mode, it is coated on nitrocellulose filter and obtains detection line I, the package amount of the every centimetre of upper required zearalenone of detection line I-bovine serum albumin(BSA) conjugate (ZEA-BSA) is 200ng; Aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is mixed with to the solution of 0.25mg/mL with coated damping fluid, position in distance detection line I3mm is coated in it on nitrocellulose filter and obtains detection line II by some spray mode, the package amount of the upper required aflatoxin B1 of every centimetre of detection line II-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 100ng, then under 37 ℃ of conditions dry 30 minutes;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with to the coating buffer of 0.25mg/mL with coated damping fluid, position in distance detection line I6mm, by a spray mode, by it, laterally be coated on nitrocellulose filter, obtain nature controlling line, on every centimetre of nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 100ng, then under 37 ℃ of conditions dry 1 hour;
Described coated damping fluid is: the 0.1g bovine serum albumin(BSA), and 0.08g sodium chloride, the 0.029g disodium hydrogen phosphate, 0.002g potassium chloride, the 0.002g potassium dihydrogen phosphate, add the water constant volume to the 10mL gained.
The long 22mm of described nitrocellulose filter, wide 4mm.
(3) preparation of sample pad
Glass fibre membrane is cut out to growth 12mm, and the specification of wide 4mm, put into confining liquid A and soak, and takes out, and under 37 ℃ of conditions, drying is 8 hours, obtains sample pad, then puts room temperature preservation in exsiccator.
Described confining liquid A is: the 2g bovine serum albumin(BSA), and 2.5g sucrose, the 0.02g sodium azide, 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate, add water and be settled to the 100mL gained;
(4) preparation of gold mark pad
Glass fibre membrane is cut out to the specification of the wide 4mm of growth 10mm, putting into confining liquid B soaks, take out, under 37 ℃ of conditions, drying is 8 hours, on dry glass fibre membrane, by a spray mode to the horizontal mixed solution of the anti-zearalenone monoclonal antibody solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of spraying nano gold mark and nano gold mark on dry glass fibre membrane, every centimetre of consumption that sprays the aspergillus flavus resisting toxin general purpose single clonal antibody of the required nano gold mark of length is 135ng, the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 300ng, then vacuum freeze drying is 2 hours, put room temperature preservation in exsiccator,
Described confining liquid B is: 2g bovine serum albumin(BSA), 2.5g sucrose, 1.6775g sodium chloride, 0.05g Tween-20,0.3g polyvinylpyrrolidone, 0.02g sodium azide, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate, add water and be settled to the 100mL gained;
The general monoclonal antibody solution of the aspergillus flavus resisting toxin of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.4mL0.1mol/L wet chemical, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirred, continue to stir 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby, wherein the mass concentration of the general monoclonal antibody solution of aspergillus flavus resisting toxin of nano gold mark is 0.04mg/mL;
The anti-zearalenone monoclonal antibody solution of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.425mL0.1mol/L wet chemical, the anti-zearalenone monoclonal antibody aqueous solution that slowly adds 2.5mL0.1mg/mL under the state stirred, continue to stir 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby, wherein the mass concentration of the anti-zearalenone monoclonal antibody solution of nano gold mark is 0.05mg/mL:
In described nano-Au solution, the particle diameter of nm of gold is 15nm;
Described 0.1mol/L wet chemical is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: the 2.0g PEG-400, and the 0.2g Sodium azide, 0.1235g boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained.
(5) assembling of test strips
Paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad in the one side of cardboard, adjacent each pad overlapping connection in junction, overlapping length is 1mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone, sees Fig. 1 and Fig. 2.
The application of the immuno-chromatographic test paper strip of above-mentioned synchronous detection aflatoxin and zearalenone composite pollution in corn sample detects
Take levigate 1#, 2# and 3# corn sample to be measured, add the methanol aqueous solution that volumetric concentration is 70%, the mass volume ratio of testing sample and methanol aqueous solution is 4g/mL, mix, under 50 ℃ of water-baths, ultrasonic extraction is 10 minutes, standing 10 minutes, by supernatant liquor, be 3 times of extract dilute with waters, the final volume concentration that makes methyl alcohol in dilution is 23.3%, obtain sample solution, get again the sample solution that 100 μ L have diluted and dropwise add a synchronous sample pad that detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution as detecting liquid, it is as test strip, get methanol aqueous solution that 100 μ L methanol concentrations are 23.3% as negative controls simultaneously, dropwise add another synchronous sample pad that detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution, it is test strips in contrast, reading result after 15 minutes.
Testing result: the nature controlling line of 1# testing sample test strip demonstrates red stripes, and detection line I and detection line II all do not develop the color, and see Fig. 3-1, and judge thus: in the 1# testing sample solution, the content of zearalenone is equal to or higher than 4ng/mL; The content of aflatoxin is equal to or higher than 1ng/mL; The content that can obtain zearalenone in the 1# testing sample through converting is equal to or higher than 48ng/g; The content of aflatoxin is equal to or higher than 12ng/g.
The nature controlling line of 2# testing sample test strip demonstrates red stripes, detection line I color is more shallow than contrast ELISA test strip line I, detection line II does not develop the color, see Fig. 3-2, judge thus: in the 2# testing sample solution, the content of zearalenone is equal to or higher than 1ng/mL and, lower than 4ng/mL, the content of aflatoxin is equal to or higher than 1ng/mL; The content can obtain zearalenone in the 2# testing sample through converting is equal to or higher than 12ng/g and lower than 48ng/g; The content of aflatoxin is equal to or higher than 12ng/g.
The nature controlling line of 3# testing sample test strip demonstrates red stripes, detection line I color in detection line I color and control stripes bar approaches, detection line II is more shallow than contrast ELISA test strip line II, see Fig. 3-3, judge thus: in the 3# testing sample solution, the content of zearalenone is lower than 1ng/mL, and the content of aflatoxin is equal to or higher than 0.25ng/mL and lower than 1ng/mL; Can obtain the content of zearalenone in the 3# testing sample lower than 12ng/g through converting; The content of aflatoxin is equal to or higher than 3ng/g, and lower than 12ng/g.
The synchronous preparation method who detects the immuno-chromatographic test paper strip of aflatoxin and two kinds of mycotoxins of zearalenone, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out to growth 18mm, and the specification of wide 3mm, obtain adsorptive pads;
(2) preparation of detecting pad
Being coated with of detection line:
Zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) is mixed with to the coating buffer of 0.3mg/mL with coated damping fluid, on the distance nitrocellulose filter along the position of 20mm, by a spray mode, it is coated on nitrocellulose filter and obtains detection line I, the package amount of the every centimetre of upper required zearalenone of detection line I-bovine serum albumin(BSA) conjugate (ZEA-BSA) is 300ng; Aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is mixed with to the solution of 0.5mg/mL with coating buffer, position in distance detection line I2mm is coated in it on nitrocellulose filter and obtains detection line II by some spray mode, the package amount of the upper required aflatoxin B1 of every centimetre of detection line II-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 300ng, then under 40 ℃ of conditions dry 30 minutes;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with to the coating buffer of 0.4mg/mL with coated damping fluid, position in distance detection line I7mm, by a spray mode, by it, laterally be coated on nitrocellulose filter, obtain nature controlling line, on every centimetre of nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50ng, then under 40 ℃ of conditions dry 1.5 hours;
Described coated damping fluid is: the 0.2g bovine serum albumin(BSA), and 0.08g sodium chloride, the 0.029g disodium hydrogen phosphate, 0.002g potassium chloride, the 0.002g potassium dihydrogen phosphate, add the water constant volume to the 10mL gained.
The long 28mm of described nitrocellulose filter, wide 3mm.
(3) preparation of sample pad
Glass fibre membrane is cut out to growth 15mm, and the specification of wide 3mm, put into confining liquid A and soak, and takes out, and under 40 ℃ of conditions, drying is 6 hours, obtains sample pad, then puts room temperature preservation in exsiccator.
Described confining liquid A is: the 2g bovine serum albumin(BSA), and 3g sucrose, the 0.05g sodium azide, 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate, add water and be settled to the 100mL gained;
(4) preparation of gold mark pad
Glass fibre membrane is cut out to growth 12mm, the specification of wide 3mm, putting into confining liquid B soaks, take out, under 40 ℃ of conditions, drying is 6 hours, on dry glass fibre membrane, by a spray mode to the horizontal mixed solution of the anti-zearalenone monoclonal antibody solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of spraying nano gold mark and nano gold mark on dry glass fibre membrane, every centimetre of consumption that sprays the aspergillus flavus resisting toxin general purpose single clonal antibody of the required nano gold mark of length is 200ng, the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 400ng, then vacuum freeze drying is 3 hours, put room temperature preservation in exsiccator,
Described confining liquid B is: 1g bovine serum albumin(BSA), 3g sucrose, 1g sodium chloride, 0.1g Tween-20,0.5g polyvinylpyrrolidone, 0.05g sodium azide, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate, add water and be settled to the 100mL gained;
The preparation method that the general monoclonal antibody solution of aspergillus flavus resisting toxin of described nano gold mark and the anti-zearalenone monoclonal antibody solution of nano gold mark are is as embodiment 2, and difference is; In its nano-Au solution used, the particle diameter of nm of gold is 20nm;
(5) assembling of test strips
Paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad in the one side of cardboard, adjacent each pad overlapping connection in junction, overlapping length is 3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone.
The application of the immuno-chromatographic test paper strip of above-mentioned synchronous detection aflatoxin and zearalenone in corn sample detects
Take levigate corn sample to be measured, add the methanol aqueous solution that volumetric concentration is 60%, the mass volume ratio of testing sample and methanol aqueous solution is 4g/mL, mix, under 50 ℃ of water-baths, ultrasonic extraction is 10 minutes, standing 10 minutes, by supernatant liquor, be 3 times of extract dilute with waters, the final volume concentration that makes methyl alcohol in dilution is 20%, obtain sample solution, get again the sample solution that 100 μ L have diluted and dropwise add a synchronous sample pad that detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution as detecting liquid, it is as test strip, get methanol aqueous solution that 100 μ L methanol concentrations are 20% as negative controls simultaneously, dropwise add another synchronous sample pad that detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution, it is test strips in contrast, reading result after 20 minutes.
Testing result: the nature controlling line of testing sample test strip demonstrates red stripes, and detection line I and detection line II all do not develop the color, and judge thus: in testing sample solution, the content of zearalenone is equal to or higher than 4ng/mL; In testing sample solution, the content of aflatoxin is equal to or higher than 1ng/mL; Can obtain the content of zearalenone in testing sample through converting and be equal to or higher than 48ng/g; The content of aflatoxin is equal to or higher than 12ng/g.
Claims (10)
1. synchronously detect the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution, it is characterized in that: it comprises cardboard, the one side of cardboard is pasted adsorptive pads from top to bottom successively, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, described detecting pad be take nitrocellulose filter as base wad, nitrocellulose filter is provided with horizontal nature controlling line and detection line, described detection line is positioned at the below of nature controlling line, number is two, be spaced apart, be coated with respectively zearalenone-bovine serum albumin(BSA) conjugate and aflatoxin B1-bovine serum albumin(BSA) conjugate on described two detection lines, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, described gold mark pad transverse jet scribbles the anti-zearalenone monoclonal antibody of aspergillus flavus resisting toxin general purpose single clonal antibody and the nano gold mark of nano gold mark, the hybridoma cell strain 1C11 secretion that described aspergillus flavus resisting toxin general purpose single clonal antibody is CCTCC NO.C201013 by deposit number produces, and the hybridoma cell strain 2D3 secretion that anti-zearalenone monoclonal antibody is CCTCC NO.C201328 by deposit number produces.
2. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution, is characterized in that: the long 16～18mm of described adsorptive pads, wide 3～4mm, the long 18～30mm of detecting pad, wide 3～4mm; Long 10～the 12mm of gold mark pad, wide 3～4mm; Long 12～the 15mm of sample pad, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm.
3. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution, it is characterized in that: the spacing on described detecting pad between two detection lines is 2-4mm, on the detection line of close nature controlling line and nitrocellulose filter, the spacing on edge is 15～20mm, and the described detection line near nature controlling line and the spacing of nature controlling line are 5～7mm.
4. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution is characterized in that: the package amount that described detecting pad is coated with every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate on the detection line of zearalenone-bovine serum albumin(BSA) conjugate is 100～300ng; On the detection line of coated aflatoxin B1-bovine serum albumin(BSA) conjugate, the package amount of every centimetre of needed aflatoxin B1-bovine serum albumin(BSA) conjugate is 100～300ng; On nature controlling line, the package amount of every centimetre of anti-mouse polyclonal antibody of needed rabbit is 50～200ng.
5. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution is characterized in that: in described gold mark pad, the particle diameter of nm of gold used is 15～20nm; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the upper every centimetre of spraying length of described gold mark pad is required is 100～200ng, and the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 200～400ng.
6. detect simultaneously and rapidly the preparation method of the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution, it is characterized in that: it comprises the following steps:
The preparation of adsorptive pads
Thieving paper is cut out and is obtained adsorptive pads;
The preparation of detecting pad
Being coated with of detection line:
The conjugate of zearalenone-bovine serum albumin(BSA) conjugate and aflatoxin B1-bovine serum albumin(BSA) is mixed with respectively to the coating buffer of 0.25～0.5mg/mL with coated damping fluid, by a spray mode, it is coated with respectively on nitrocellulose filter, obtain two detection lines, then under 37～40 ℃ of conditions dry 30～60 minutes; On the detection line of described coated zearalenone-bovine serum albumin(BSA) conjugate, the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate is 100～300ng; On the detection line of coated aflatoxin B1-bovine serum albumin(BSA) conjugate, the package amount of needed aflatoxin B1-bovine serum albumin(BSA) conjugate is 100～300ng, spacing between described two detection lines is 2-4mm, and on the detection line of close nature controlling line and nitrocellulose filter, the spacing on edge is 15～20mm;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with to the solution of 0.2～0.4mg/mL with coated damping fluid, position in the detection line 5～7mm apart near nature controlling line, by a spray mode, by it, laterally be coated on nitrocellulose filter, obtain nature controlling line, on every centimetre of nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50～200ng, then under 37～40 ℃ of conditions dry 1～2 hour;
The preparation of sample pad
Glass fibre membrane is put into to confining liquid and soak, take out, under 37～40 ℃ of conditions, drying is 6～10 hours, obtains sample pad, then puts room temperature preservation in exsiccator;
The preparation of gold mark pad
Glass fibre membrane is put into to confining liquid to soak, take out, under 37～40 ℃ of conditions, drying is 6～10 hours, by a spray mode to the horizontal mixed solution of the anti-zearalenone monoclonal antibody solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of spraying nano gold mark and nano gold mark on dry glass fibre membrane, wherein: every centimetre of consumption that sprays the aspergillus flavus resisting toxin general purpose single clonal antibody of the required nano gold mark of length is 100～200ng, the consumption of the anti-zearalenone monoclonal antibody of required nano gold mark is 200～400ng, then vacuum freeze drying is 2～4 hours, put room temperature preservation in exsiccator, the hybridoma cell strain 1C11 secretion that described aspergillus flavus resisting toxin general purpose single clonal antibody is CCTCC NO.C201013 by deposit number produces, and the hybridoma cell strain 2D3 secretion that described anti-zearalenone monoclonal antibody is CCTCC NO.C201328 by deposit number produces,
The assembling of test strips
Paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad in the one side of cardboard, adjacent each pad overlapping connection in junction, overlapping length is 1～3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone composite pollution.
7. the preparation method who detects simultaneously and rapidly the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution according to claim 6, it is characterized in that: in the every 10mL of described coated damping fluid, contain: bovine serum albumin(BSA) 0.1～0.2g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g.
8. the preparation method who detects simultaneously and rapidly the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution according to claim 6, it is characterized in that: in the every 100mL of the confining liquid used in the preparation process of described sample pad, contain: bovine serum albumin(BSA) 1～2g, sucrose 2～3g, sodium azide 0.02～0.05g, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g;
In the every 100mL of confining liquid used in the preparation process of described gold mark pad, contain: bovine serum albumin(BSA) 1～2g, sucrose 2～3g, sodium chloride 1～2g, Tween-20 0.05～0.1g, polyvinylpyrrolidone 0.2～0.5g, sodium azide 0.02～0.05g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
9. the preparation method who detects simultaneously and rapidly the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution according to claim 6, it is characterized in that: the general monoclonal antibody solution of the aspergillus flavus resisting toxin of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.4mL0.1mol/L wet chemical, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirred, continue to stir 30min, to add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min, after 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation, by the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid, again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with the mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby,
The anti-zearalenone monoclonal antibody solution of described nano gold mark adopts unsaturated labelling method to prepare, its concrete grammar is: get the nano-Au solution that the commercially available mass concentration of 50.0mL is 0.01%, regulate the pH value with the 0.425mL0.1mol/L wet chemical, the anti-zearalenone monoclonal antibody aqueous solution that slowly adds 2.5mL0.1mg/mL under the state stirred, continue to stir 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, get supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, add the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that to preserve liquid with the mark washing resuspended, obtain the 5.0mL concentrate, put 4 ℃ of refrigerators standby;
Described 0.1mol/L wet chemical is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: the 2.0g PEG-400, and the 0.2g Sodium azide, 0.1235g boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained.
10. according to the application of the immuno-chromatographic test paper strip of the described synchronous detection aflatoxin of any one in claim 1-5 and zearalenone composite pollution, it is characterized in that: its application process is: take levigate testing sample, add the methanol aqueous solution that volumetric concentration is 60～80%, mix, under 50～60 ℃ of water-baths, ultrasonic extraction is 5～10 minutes, standing 5～10 minutes, by supernatant liquor, it is the extract dilute with water, the final volume concentration that makes methyl alcohol in dilution is 20～30%, obtain testing sample solution, get again this testing sample solution of 80-150 μ L as being detected on the sample pad of immuno-chromatographic test paper strip that detects liquid and dropwise join synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, separately get the consistent methanol aqueous solution of isopyknic methanol concentration as negative controls, dropwise add on the sample pad of another immuno-chromatographic test paper strip that synchronously detects aflatoxin and zearalenone composite pollution, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar: when on test strip, the coated zearalenone-detection line color of bovine serum albumin(BSA) conjugate approaches with the color of corresponding detection line on the control stripes bar, show in testing sample solution that zearalenone content is lower than 1ng/mL, during than corresponding detection line of light color, show in testing sample solution that zearalenone content is equal to or higher than 1ng/mL and lower than 4ng/mL, while not developing the color, show that in testing sample solution, the content of zearalenone is equal to or higher than 4ng/mL,
When the detection line color of the conjugate of coated aflatoxin B1-bovine serum albumin(BSA) approaches with the color of corresponding detection line on the control stripes bar on test strip, show in testing sample solution that aflatoxin content is lower than 0.25ng/mL; During than corresponding detection line of light color, show in testing sample solution that aflatoxin content is equal to or higher than 0.25ng/mL and lower than 1ng/mL; While not developing the color, show that in testing sample solution, the content of aflatoxin is equal to or higher than 1ng/mL;
When nature controlling line does not develop the color, no matter whether the detection line of test strip develops the color, and it is invalid that this test strips is judged to;
Finally by converting and obtaining the content of aflatoxin and zearalenone in testing sample.
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