CN103257230A - 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 detect synchronously 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 that is present in the cereal, are the secondary metabolites that is produced by the fungi that pollutes cereal.Grain and feed are not too high by temperature or humidity in abundant drying or the transporting procedures in the results process, all may breed for fungi growth suitable condition is provided, thereby cause the pollution of mycotoxin.Aflatoxin can destroy the liver organization of humans and animals strongly, can cause liver cancer even death when serious; Zearalenone has the class estrogen action, can cause the animal acute and chronic poisoning, causes animal reproduction parasthenia even death, and these two kinds of toxin are prevalent in grain and the feed, and human beings'health and safety are caused serious threat.In view of the damaging effect of these two kinds of mycotoxins and the extensive generation in grain and feed thereof, the content of these two kinds of mycotoxins has carried out strict restriction in countries in the world grain and the feed.In order to strengthen the detection to these mycotoxins in grain and the feed, ensure people's consumption safety, exploitation is at detection technique, the especially fast detecting of these mycotoxins, be to understand and grasp food and feed safety health information, strengthen the important step of food security consumption.
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, all can carry out in common laboratory, but detect that reagent dosage is big, complex operation, other component serious interference, poor accuracy, can not be accurately quantitative, and bigger 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 height of the sample that requirement detects, sample pretreatment process is loaded down with trivial details, length consuming time requires height to experimental situation and testing staff, is difficult to realize fast detecting, detection cost height is not suitable for field quick detection.Immune analysis method has overcome the shortcoming of thin layer chromatography and instrumental method because 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 advantage such as on-the-spot batch detection and obtained fast development in recent years.Based on the immunochromatography technique of colloid gold label antibody and antigentic specificity association reaction since its testing result naked eyes as seen, do not need large-scale instrument and equipment, the detection cost is low, analysis time is short, and qualitative, online, the fast detecting at minimal residue things such as mycotoxins obtained widespread use in recent years.Yet the existing colloidal gold immuno-chromatography test paper strip that detects for mycotoxin mostly can only detect a kind of mycotoxin.And the planting patterns of China smallholder decentralized, mycotoxin incidence height in the agricultural product, and same agricultural product are subjected to multiple mycotoxin contamination of heavy big, therefore press for the detection technique that can detect multiple mycotoxin synchronously, to realize synchronous, the fast monitored to mycotoxin composite pollution in grain and the feed etc.
Summary of the invention
Problem to be solved by this invention provides 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 the 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:
Detect the immuno-chromatographic test paper strip (seeing Fig. 1 and Fig. 2) of aflatoxin and zearalenone composite pollution synchronously, 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 the junction, described detecting pad is base wad with the nitrocellulose filter, 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 zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) on described two detection lines respectively, 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; Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 secretion generation of CCTCC NO. C201013 by deposit number, and anti-zearalenone monoclonal antibody is the hybridoma cell strain 2D3 secretion generation of CCTCC NO. C201328 by deposit number; This hybridoma cell strain 2D3 has been preserved in Chinese typical 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 the described detecting pad between two detection lines is 2-4mm, is 15～20mm near the detection line of nature controlling line with the spacing of nitrocellulose filter upper edge, and the detection line of described close nature controlling line and the spacing of nature controlling line are 5～7mm.
Press such scheme, described detecting pad bag is 100～300ng by the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) on the detection line of zearalenone-bovine serum albumin(BSA) conjugate; Bag is 100～300ng by the package amount of every centimetre of needed aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) on the detection line of aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA); The package amount of every centimetre of anti-mouse polyclonal antibody of needed rabbit is 50～200ng on the nature controlling line.
Press such scheme, the particle diameter of used nm of gold is 15～20nm in the described gold mark pad; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the last 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 the preparation method of the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution as mentioned above simultaneously and rapidly, may further comprise the steps:
(1) preparation of adsorptive pads
Thieving paper cut out namely get adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate (AFB1-BSA) of zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and aflatoxin B1-bovine serum albumin(BSA) is cushioned the coating buffer that liquid is mixed with 0.25～0.5mg/mL respectively with bag, with a spray mode it is wrapped quilt respectively on nitrocellulose filter, obtain two detection lines, under 37～40 ℃ of conditions dry 30～60 minutes then; Described bag is 100～300ng by the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) on the detection line of zearalenone-bovine serum albumin(BSA) conjugate; Bag is 100～300ng by the package amount of needed aflatoxin B1 on the detection line of aflatoxin B1-bovine serum albumin(BSA) conjugate-bovine serum albumin(BSA) conjugate (AFB1-BSA), spacing between described two detection lines is 2-4mm, is 15～20mm near the detection line of nature controlling line and the spacing of nitrocellulose filter upper edge;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned the solution that liquid is mixed with 0.2～0.4mg/mL with bag, in the position of distance near the detection line 5～7mm of nature controlling line, with a spray mode it is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50～200ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 1～2 hour then;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid soak, take out, drying is 6～10 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then;
(4) preparation of gold mark pad
Glass fibre membrane is put into confining liquid to soak, take out, drying is 6～10 hours under 37～40 ℃ of conditions, with a spray mode horizontal mixed solution of the anti-zearalenone monoclonal anti liquid solution of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of spraying nano gold mark and nano gold mark on the 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, vacuum freeze drying is 2～4 hours then, puts room temperature preservation in the exsiccator; Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 secretion generation of CCTCC NO.C201013 by deposit number, and described anti-zearalenone monoclonal antibody is the hybridoma cell strain 2D3 secretion generation of CCTCC NO.C201328 by deposit number;
(5) assembling of test strips
Paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlapping connection in the junction, overlapping length is 1～3mm, namely gets the immuno-chromatographic test paper strip that detects aflatoxin and zearalenone composite pollution synchronously.
Press such scheme, described bag is cushioned among the every 10mL of liquid and contains: 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 among 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, contain among the every 100mL of confining liquid that described step (4) is used: 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 aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, and it is standby to put 4 ℃ of refrigerators;
The anti-zearalenone monoclonal anti liquid solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, and it is standby to put 4 ℃ of refrigerators;
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: 2.0g polyglycol-20000, 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 aforesaid synchronous detection aflatoxin and zearalenone composite pollution, method is as follows: take by weighing levigate testing sample, the adding volumetric concentration is 60～80% methanol aqueous solution, mixing, ultrasonic extraction is 5～10 minutes under 50～60 ℃ of water-baths, left standstill 5～10 minutes, be the extract dilute with water with supernatant liquor, the final volume concentration that makes methyl alcohol in the dilution is 20～30%, obtain testing sample solution, getting this testing sample solution of 80-150 μ L again detects as detecting on the sample pad of immuno-chromatographic test paper strip that liquid dropwise joins synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, other gets the methanol aqueous solution of isopyknic methanol concentration unanimity as negative controls, dropwise add on the sample pad of another immuno-chromatographic test paper strip that detects aflatoxin and zearalenone composite pollution synchronously, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar: when bag on the test strip by the color of corresponding detection line on the detection line color of zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and the control stripes bar near the time, show that zearalenone content is lower than 1ng/mL in the testing sample solution; During than corresponding detection line of light color, show that zearalenone content is equal to or higher than 1ng/mL and is lower than 4ng/mL in the testing sample solution; When not developing the color, show that the content of zearalenone is equal to or higher than 4ng/mL in the testing sample solution;
When bag on the test strip by the color of corresponding detection line on the detection line color of the conjugate of aflatoxin B1-bovine serum albumin(BSA) (AFB1-BSA) and the control stripes bar near the time, show that aflatoxin content is lower than 0.25ng/mL in the testing sample solution; During than corresponding detection line of light color, show that aflatoxin content is equal to or higher than 0.25ng/mL and is lower than 1ng/mL in the testing sample solution; When not developing the color, show that the content of aflatoxin is equal to or higher than 1ng/mL in the testing sample solution;
When nature controlling line did not develop the color, no matter whether the detection line of test strip developed the color, and it is invalid that this test strips is judged to,
Namely get the content of aflatoxin and zearalenone in the testing sample finally by converting.
The principle of work of this immuno-chromatographic test paper strip in aflatoxin and zearalenone composite pollution detect synchronously: 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 moved 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 were dissolved.When containing aflatoxin in the sample, the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that aflatoxin will fill up with the gold mark is in conjunction with also together upwards swimming, when aflatoxin B1-bovine serum albumin(BSA) conjugate detection of antigens line is being fixed in its arrival, 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, aflatoxin content is more high in the sample, antigen on the detection line can in conjunction with the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark will be more few, more shallow in the colour developing band color that detection line forms; When containing zearalenone in the sample, the anti-zearalenone monoclonal antibody of the nano gold mark that zearalenone will fill up with the gold mark is in conjunction with also together upwards swimming, when zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) detection of antigens line I is being fixed in its arrival, antigen will be competed limited antigen binding site on the anti-zearalenone monoclonal antibody of combining nano gold mark with zearalenone, zearalenone content is more high in the sample, antigen on the detection line can in conjunction with the anti-zearalenone monoclonal antibody of nano gold mark will be more few, more shallow in the colour developing band color that detection line forms.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 whether contain this two kinds of mycotoxins in the sample, the antibody of the antibody of the antimycotic toxin of the nano gold mark that the antigen on the not tested survey line is intercepted and captured or the antimycotic toxin of nano gold mark and the bond of mycotoxin are combined and are developed the color by enrichment continuing to move to nature controlling line and the anti-mouse polyclonal antibody of the rabbit on nature controlling line.Accordingly, respectively with bag on the test strip by the detection line of the conjugate of aflatoxin B1-bovine serum albumin(BSA) and bag by the contrast that develops the color of corresponding detection line color on the detection line of zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) and the control stripes bar, can obtain the composite pollution situation of aflatoxin and these two kinds of mycotoxins of zearalenone in the sample.
Beneficial effect of the present invention:
(1) one goes on foot, detects simultaneously aflatoxin and zearalenone.Immuno-chromatographic test paper strip provided by the invention can be in synchronous, the fast detecting of a test strips realization to aflatoxin and two kinds of mycotoxins of zearalenone, the antibody that uses is monoclonal antibody, specificity is good, highly sensitive, each mycotoxin is noiseless between detecting, and is 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 the solution, and the lowest detection of zearalenone is limited to 1ng/mL, and this detectability can satisfy European Union to the requirement of limiting the quantity of of these two kinds of mycotoxins in the food.
(3) simple to operate.Sample pre-treatments only need leave standstill ultrasonic extraction in the methanol-water extract adding sample 5～10 minutes 5～10 minutes then, got the supernatant dilution and can detect, and whole sample pre-treatment process is simple, quick.Description of drawings
Fig. 1 is the front elevation of the immuno-chromatographic test paper strip of synchronous detection aflatoxin of the present invention and zearalenone composite pollution;
Fig. 2 is the side view of the immuno-chromatographic test paper strip of synchronous detection aflatoxin of the present invention and zearalenone composite pollution;
Fig. 3 is the process decision chart as a result of embodiment 2;
Among the figure: 1 cardboard, 2 adsorptive pads; 3 detecting pads; 4 gold medals mark pad; 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. aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 secretion generation of CCTCC NO.C201018 by deposit number, be that reported method makes in advance in the patent of CN201010245095.5 according to number of patent application specifically, the preparation method is: hybridoma cell strain 1C11 is injected the BALB/c mouse of handling with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt sad-ammonium sulfate method antibody purification, concrete operations are: filter mouse ascites with double-deck filter paper, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stir and slowly add caprylic acid down, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min, abandon precipitation, after the supernatant that obtains filtered with double-deck filter paper, the volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffer, 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 ℃ leave standstill 2h, 4 ℃ then, 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, to the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, use the freeze drier freeze-drying afterwards, collect freeze-dried powder, namely get the good aspergillus flavus resisting toxin general purpose single clonal antibody of purifying, antibody is placed-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 adds water and is settled to the 100mL gained.
B. anti-zearalenone monoclonal antibody is the hybridoma cell strain 2D3 generation of CCTCC NO.C201328 by deposit number, and its concrete preparation method is:
Hybridoma cell strain 2D3 is injected the BALB/c mouse of handling with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt sad-ammonium sulfate method antibody purification, the concrete operations step is: filter mouse ascites with double-deck filter paper, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stir and slowly add caprylic acid down, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min, abandon precipitation, after the supernatant that obtains filtered with double-deck filter paper, the volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffer, with the pH value to 7.4 that the sodium hydroxide solution of 2mol/L is regulated this mixed liquor, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min abandons supernatant, with the 0.01mol/L of gained precipitation with former ascites volume 1/10, the pH value is that 7.4 phosphate buffer is resuspended, the bag filter of packing into, to the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, use the freeze drier freeze-drying afterwards, collect freeze-dried powder, namely get the good anti-zearalenone monoclonal antibody of purifying, antibody is placed-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 adds 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 adds water and is 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 tiring of antibody that records the BALB/c mouse ascites purifying acquisition of injection hybridoma cell strain 2D3 with the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method can reach 1.5 * 10
5, i.e. anti-zearalenone monoclonal antibody dilution 1.5 * 10
5Times the time measured in solution result positive.Identify that with 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: above-mentioned hybridoma cell strain 2D3 obtains according to following method screening:
(1) animal immune
6 of purchase BALB/c mouse in 6 age in week, the zearalenone comlete antigen ZEA-BSA that immunity is commercially available.Immunity is with 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 for the second time being immune to for 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 immunity for the second time be 4 weeks at interval, and immunization ways is identical with it, are immune to for the third time for the 4th time to carry out after immune 3 weeks, and immunization ways is immune identical with the second time, is similarly lumbar injection.4 times immunizing dose is identical, is every mouse 100 μ g.Back 8 days of each immunity 3 times, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum to tire.Back 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 with the indirect competitive ELISA method, selection is tired, sensitivity all the mouse of higher relatively serum correspondence carry out last booster immunization, immunizing dose is 2 times of front.Zearalenone comlete antigen ZEA-BSA purchases the company in German aokin.
(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 Fusion of Cells according to a conventional method, concrete steps: kill immune mouse under the aseptic condition, 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, merge with 50%PEG, merged 1 minute, and slowly added the RPMI-1640 basic culture solution then, centrifugal, remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with the cell complete medium that 20mL contains 1%HAT, and the cell that has hanged is joined in the 80mL semisolid culturemedium, is added to behind the mixing on the 6 porocyte culture plates, 1.5mL/ the hole places 37 ℃ of CO2gas incubator to cultivate.The cell complete medium of the described 1%HAT of containing contains 20% (percent by volume) hyclone, 75% (percent by volume) RPMI-1640 basic culture solution, 1% (percent by weight) L-glutamine, 1% (percent by volume) HEPES, 1% (percent by volume) two anti-(the every ml penicillin of 10000 units 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 for containing the cell complete medium of 1% (mass percent) methylcellulose; RPMI-1640 basic culture solution, HEPES, two anti-and L-glutamine are purchased the company in Hyclone; 1% hypoxanthine-aminopterin-thymidine is that HAT and methylcellulose are purchased the company in Sigma-Aldrich.
(3) screening of cell line and clone
Treat 2-3 week after the Fusion of Cells, when cell colony length is visible to people's naked eyes, to clone from this nutrient culture media with micropipettor and to draw, moving to 96 porocyte culture plates adopts liquid to amplify cultivation, every hole moves into 1 clone, treat that cell grows at the bottom of the full hole at 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, namely carry out antibody test.Adopt the ELISA method that the culture hole that the hybridoma growth is arranged is screened, 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; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected, former as competition with zearalenone, all (the higher finger competition of light absorption value was that 0 hole is that the final measured value in positive control hole is higher originally, and the higher finger inhibiting rate of sensitivity is 50% o'clock competition original content that is IC higher hole to select light absorption value and sensitivity
50Be worth less), adopt limiting dilution assay to carry out subclone, adopt same two-step approach to detect behind the subclone, so repeat subclone 2-3 time after, acquisition hybridoma cell strain 2D3.
Hybridoma cell strain 2D3 antibody variable region sequencing
(1) extracts total RNA: adopt day total RNA extraction reagent box of root company and extract total RNA that can produce hybridoma cell strain 2D3 to specifications;
(2) synthetic cDNA: the total RNA that obtains with step 1 is template, oligo (dT)
15Be primer, according to SuperScript
TM-2II reverse transcriptase instructions carries out reverse transcription, synthetic cDNA first chain; Primer oligo (dT)
15Buied by Invitrogen;
(3) PCR method clone variable region gene: according to the conservative site design primer of the medium and small mouse antibody genes sequence of GENEBANK, be light, the heavy chain variable region gene of masterplate amplification antibody with cDNA.The PCR program is: 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.After the agarose gel electrophoresis separation of PCR product through 1% (percent by weight), reclaim dna fragmentation with the kit purifying, be connected among the carrier pMD18-T transformed into escherichia coli DH5 α competent cell, the picking positive colony is delivered to Shanghai Sani's bio tech ltd and is checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is that (22mer) (32mer) wherein S, M, R and W are the merger base to 5 '-AGG TSM ARC TGC AGS AGT CWG G-3 ' with 5 '-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3 ', M=A/C, R=A/G, S=C/G, W=A/T, 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 who obtains: the long 315bp of variable region of heavy chain coding gene sequence, sequence is shown in SEQ ID NO:1, derive the coded variable region of heavy chain of this gene order according to the gene order that obtains and be made up of 105 amino acid, sequence is shown in SEQ ID NO:3.The long 329bp of variable region of light chain coding gene sequence, sequence is derived the coded variable region of light chain of this gene order according to the gene order that obtains and is made up of 109 amino acid shown in SEQ ID NO:2, and sequence is shown in SEQ IDNO:4.
Detect the preparation method of the immuno-chromatographic test paper strip of aflatoxin and two kinds of mycotoxins of zearalenone synchronously, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out growth 16mm, and the specification of wide 4mm namely gets adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
Zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) is cushioned the coating buffer that liquid is mixed with 0.4mg/mL with bag, position in distance nitrocellulose filter upper edge 15mm, with a spray mode its bag is obtained detection line I on nitrocellulose filter, the package amount of the every centimetre of last 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 cushioned the solution that liquid is mixed with 0.25mg/mL with bag, position in distance detection line I3mm is obtained detection line II with some spray mode with its bag on nitrocellulose filter, the package amount that every centimetre of detection line II goes up required aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 100ng, under 37 ℃ of conditions dry 30 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned the coating buffer that liquid is mixed with 0.25mg/mL with bag, position in distance detection line I6mm, with a spray mode it is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 100ng on every centimetre of nature controlling line, under 37 ℃ of conditions dry 1 hour then;
Described bag is cushioned liquid: 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 adds 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 growth 12mm, and the specification of wide 4mm is put into confining liquid A and is soaked, and takes out, and drying is 8 hours under 37 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then.
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 adds water and is settled to the 100mL gained;
(4) preparation of gold mark pad
Glass fibre membrane is cut out the specification of the wide 4mm of growth 10mm, putting into confining liquid B soaks, take out, drying is 8 hours under 37 ℃ of conditions, on dry glass fibre membrane, with a spray mode horizontal mixed solution of the anti-zearalenone monoclonal anti liquid solution of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of spraying nano gold mark and nano gold mark on the 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, vacuum freeze drying is 2 hours then, puts room temperature preservation in the 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 adds water and is settled to the 100mL gained;
The aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
The anti-zearalenone monoclonal anti liquid solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the anti-zearalenone monoclonal anti liquid solution of nano gold mark is 0.05mg/mL:
The particle diameter of nm of gold is 15nm in the described nano-Au solution;
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: 2.0g polyglycol-20000, 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 adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlapping connection in the junction, overlapping length is 1mm, namely gets the immuno-chromatographic test paper strip that detects aflatoxin and zearalenone synchronously, 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 by weighing levigate 1#, 2# and 3# corn sample to be measured, the adding volumetric concentration is 70% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 4g/mL, mixing, ultrasonic extraction is 10 minutes under 50 ℃ of water-baths, left standstill 10 minutes, be 3 times of extract dilute with waters with supernatant liquor, the final volume concentration that makes methyl alcohol in the dilution is 23.3%, obtain sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the immuno-chromatographic test paper strip of a synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, get 100 μ L methanol concentrations simultaneously and be 23.3% methanol aqueous solution as negative controls, dropwise add the sample pad that another detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution synchronously, it is test strips in contrast, reads the 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: the content of zearalenone is equal to or higher than 4ng/mL in the 1# testing sample solution; The content of aflatoxin is equal to or higher than 1ng/mL; The content that can get zearalenone in the 1# testing sample is equal to or higher than 48ng/g through converting; 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 test strips detection line I, detection line II does not develop the color, see Fig. 3-2, judge thus: the content of zearalenone is equal to or higher than 1ng/mL and is lower than 4ng/mL in the 2# testing sample solution, and the content of aflatoxin is equal to or higher than 1ng/mL; The content that can get zearalenone in the 2# testing sample is equal to or higher than 12ng/g and is lower than 48ng/g through converting; 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 the control stripes bar approaches, detection line II is more shallow than contrast test strips detection line II, see Fig. 3-3, judge thus: the content of zearalenone is lower than 1ng/mL in the 3# testing sample solution, and the content of aflatoxin is equal to or higher than 0.25ng/mL and is lower than 1ng/mL; The content that can get zearalenone in the 3# testing sample is lower than 12ng/g through converting; The content of aflatoxin is equal to or higher than 3ng/g, and is lower than 12ng/g.
Detect the preparation method of the immuno-chromatographic test paper strip of aflatoxin and two kinds of mycotoxins of zearalenone synchronously, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out growth 18mm, and the specification of wide 3mm namely gets adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
Zearalenone-bovine serum albumin(BSA) conjugate (ZEA-BSA) is cushioned the coating buffer that liquid is mixed with 0.3mg/mL with bag, position in distance nitrocellulose filter upper edge 20mm, with a spray mode its bag is obtained detection line I on nitrocellulose filter, the package amount of the every centimetre of last 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 the solution of 0.5mg/mL with coating buffer, position in distance detection line I2mm is obtained detection line II with some spray mode with its bag on nitrocellulose filter, the package amount that every centimetre of detection line II goes up required aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 300ng, under 40 ℃ of conditions dry 30 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned the coating buffer that liquid is mixed with 0.4mg/mL with bag, position in distance detection line I7mm, with a spray mode it is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50ng on every centimetre of nature controlling line, under 40 ℃ of conditions dry 1.5 hours then;
Described bag is cushioned liquid: 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 adds 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 growth 15mm, and the specification of wide 3mm is put into confining liquid A and is soaked, and takes out, and drying is 6 hours under 40 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then.
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 adds water and is settled to the 100mL gained;
(4) preparation of gold mark pad
Glass fibre membrane is cut out growth 12mm, the specification of wide 3mm, putting into confining liquid B soaks, take out, drying is 6 hours under 40 ℃ of conditions, on dry glass fibre membrane, with a spray mode horizontal mixed solution of the anti-zearalenone monoclonal anti liquid solution of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of spraying nano gold mark and nano gold mark on the 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, vacuum freeze drying is 3 hours then, puts room temperature preservation in the 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 adds water and is settled to the 100mL gained;
Preparation method such as embodiment 2 that the anti-zearalenone monoclonal anti liquid solution of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark and nano gold mark is, difference is; The particle diameter of nm of gold is 20nm in its employed nano-Au solution;
(5) assembling of test strips
Paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlapping connection in the junction, overlapping length is 3mm, namely gets the immuno-chromatographic test paper strip that detects aflatoxin and zearalenone synchronously.
The application of the immuno-chromatographic test paper strip of above-mentioned synchronous detection aflatoxin and zearalenone in corn sample detects
Take by weighing levigate corn sample to be measured, the adding volumetric concentration is 60% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 4g/mL, mixing, ultrasonic extraction is 10 minutes under 50 ℃ of water-baths, left standstill 10 minutes, be 3 times of extract dilute with waters with supernatant liquor, the final volume concentration that makes methyl alcohol in the dilution is 20%, obtain sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the immuno-chromatographic test paper strip of a synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, get 100 μ L methanol concentrations simultaneously and be 20% methanol aqueous solution as negative controls, dropwise add the sample pad that another detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution synchronously, it is test strips in contrast, reads the 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: the content of zearalenone is equal to or higher than 4ng/mL in the testing sample solution; The content of aflatoxin is equal to or higher than 1ng/mL in the testing sample solution; Can get through converting that the content of zearalenone is equal to or higher than 48ng/g in the testing sample; The content of aflatoxin is equal to or higher than 12ng/g.
Claims (10)
1. detect the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution synchronously, 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 the junction, described detecting pad is base wad with the nitrocellulose filter, 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 zearalenone-bovine serum albumin(BSA) conjugate and aflatoxin B1-bovine serum albumin(BSA) conjugate on described two detection lines respectively, 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; Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 secretion generation of CCTCC NO.C201013 by deposit number, and anti-zearalenone monoclonal antibody is the hybridoma cell strain 2D3 secretion generation of CCTCC NO.C201328 by deposit number.
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 the described detecting pad between two detection lines is 2-4mm, the detection line of close nature controlling line and the spacing of nitrocellulose filter upper edge are 15～20mm, and described is 5～7mm near the detection line of nature controlling line and the spacing of nature controlling line.
4. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution, it is characterized in that: described detecting pad bag is 100～300ng by the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate on the detection line of zearalenone-bovine serum albumin(BSA) conjugate; Bag is 100～300ng by the package amount of every centimetre of needed aflatoxin B1-bovine serum albumin(BSA) conjugate on the detection line of aflatoxin B1-bovine serum albumin(BSA) conjugate; The package amount of every centimetre of anti-mouse polyclonal antibody of needed rabbit is 50～200ng on the nature controlling line.
5. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and zearalenone composite pollution is characterized in that: the particle diameter of used nm of gold is 15～20nm in the described gold mark pad; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the last 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 the preparation method of the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution simultaneously and rapidly, it is characterized in that: it may further comprise the steps:
The preparation of adsorptive pads
Thieving paper cut out namely get adsorptive pads;
The preparation of detecting pad
The bag quilt of detection line:
The conjugate of zearalenone-bovine serum albumin(BSA) conjugate and aflatoxin B1-bovine serum albumin(BSA) is cushioned the coating buffer that liquid is mixed with 0.25～0.5mg/mL respectively with bag, with a spray mode it is wrapped quilt respectively on nitrocellulose filter, obtain two detection lines, under 37～40 ℃ of conditions dry 30～60 minutes then; Described bag is 100～300ng by the package amount of every centimetre of needed zearalenone-bovine serum albumin(BSA) conjugate on the detection line of zearalenone-bovine serum albumin(BSA) conjugate; Bag is 100～300ng by the package amount of needed aflatoxin B1-bovine serum albumin(BSA) conjugate on the detection line of aflatoxin B1-bovine serum albumin(BSA) conjugate, spacing between described two detection lines is 2-4mm, is 15～20mm near the detection line of nature controlling line and the spacing of nitrocellulose filter upper edge;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned the solution that liquid is mixed with 0.2～0.4mg/mL with bag, in the position of distance near the detection line 5～7mm of nature controlling line, with a spray mode it is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 50～200ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 1～2 hour then;
The preparation of sample pad
Glass fibre membrane is put into confining liquid soak, take out, drying is 6～10 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then;
The preparation of gold mark pad
Glass fibre membrane is put into confining liquid to soak, take out, drying is 6～10 hours under 37～40 ℃ of conditions, with a spray mode horizontal mixed solution of the anti-zearalenone monoclonal anti liquid solution of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of spraying nano gold mark and nano gold mark on the 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, vacuum freeze drying is 2～4 hours then, puts room temperature preservation in the exsiccator; Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 secretion generation of CCTCC NO.C201013 by deposit number, and described anti-zearalenone monoclonal antibody is the hybridoma cell strain 2D3 secretion generation of CCTCC NO.C201328 by deposit number;
The assembling of test strips
Paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlapping connection in the junction, overlapping length is 1～3mm, namely gets the immuno-chromatographic test paper strip that detects aflatoxin and zearalenone composite pollution synchronously.
7. the preparation method who detects the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution simultaneously and rapidly according to claim 6, it is characterized in that: described bag is cushioned among the every 10mL of liquid and contains: 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 the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution simultaneously and rapidly according to claim 6, it is characterized in that: contain among 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;
Contain among the every 100mL of confining liquid that described step (4) is used: 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 the immuno-chromatographic test paper strip of aflatoxin and zearalenone composite pollution simultaneously and rapidly according to claim 6, it is characterized in that: the aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, and it is standby to put 4 ℃ of refrigerators;
The anti-zearalenone monoclonal anti liquid solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, 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 that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 40.0mL mark and preserves 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, and it is standby to put 4 ℃ of refrigerators;
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: 2.0g polyglycol-20000, 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 each described synchronous detection aflatoxin and zearalenone composite pollution among the claim 1-5, it is characterized in that: its application process is: take by weighing levigate testing sample, the adding volumetric concentration is 60～80% methanol aqueous solution, mixing, ultrasonic extraction is 5～10 minutes under 50～60 ℃ of water-baths, left standstill 5～10 minutes, be the extract dilute with water with supernatant liquor, the final volume concentration that makes methyl alcohol in the dilution is 20～30%, obtain testing sample solution, getting this testing sample solution of 80-150 μ L again detects as detecting on the sample pad of immuno-chromatographic test paper strip that liquid dropwise joins synchronous detection aflatoxin and zearalenone composite pollution, it is as test strip, other gets the methanol aqueous solution of isopyknic methanol concentration unanimity as negative controls, dropwise add on the sample pad of another immuno-chromatographic test paper strip that detects aflatoxin and zearalenone composite pollution synchronously, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar: when bag on the test strip by the color of corresponding detection line on the detection line color of zearalenone-bovine serum albumin(BSA) conjugate and the control stripes bar near the time, show that zearalenone content is lower than 1ng/mL in the testing sample solution; During than corresponding detection line of light color, show that zearalenone content is equal to or higher than 1ng/mL and is lower than 4ng/mL in the testing sample solution; When not developing the color, show that the content of zearalenone is equal to or higher than 4ng/mL in the testing sample solution;
When bag on the test strip by the color of corresponding detection line on the detection line color of the conjugate of aflatoxin B1-bovine serum albumin(BSA) and the control stripes bar near the time, show that aflatoxin content is lower than 0.25ng/mL in the testing sample solution; During than corresponding detection line of light color, show that aflatoxin content is equal to or higher than 0.25ng/mL and is lower than 1ng/mL in the testing sample solution; When not developing the color, show that the content of aflatoxin is equal to or higher than 1ng/mL in the testing sample solution;
When nature controlling line did not develop the color, no matter whether the detection line of test strip developed the color, and it is invalid that this test strips is judged to;
Namely get the content of aflatoxin and zearalenone in the testing sample finally by converting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310115725.0A CN103257230B (en) | 2013-04-03 | 2013-04-03 | Immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044346A (en) * | 2015-06-25 | 2015-11-11 | 浙江大学 | Quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and preparation method of quantum dot fluorescence immunochromatography test strip |
| CN105334319A (en) * | 2015-10-21 | 2016-02-17 | 黑龙江省乳品工业技术开发中心 | Test strip for detecting zearalonone in milk |
| CN106977600A (en) * | 2017-03-07 | 2017-07-25 | 中国农业科学院油料作物研究所 | Purify fumonisin B1, aflatoxin, ochratoxin A, zearalenone immunosorbent and compound affinity column |
| WO2018121677A1 (en) * | 2016-12-31 | 2018-07-05 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatography kit for simultaneous detection of mixed pollutant of aflatoxin and carbaryl, preparation method therefor, and application thereof |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN110618274A (en) * | 2019-09-04 | 2019-12-27 | 山东绿都生物科技有限公司 | Chimeric ELISA kit for simultaneously and quantitatively detecting total amount of aflatoxin and zearalenone in edible oil |
| CN110806476A (en) * | 2019-11-15 | 2020-02-18 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting sickle knife fungus enol pollution of ribes diacetylenii, preparation method and application thereof |
| CN111007247A (en) * | 2019-11-15 | 2020-04-14 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatographic test strip for synchronously detecting ribes diacetate sickle-knife fungus enol, deoxynivalenol and T-2 toxin |
| CN112305216A (en) * | 2020-10-26 | 2021-02-02 | 杭州电子科技大学 | Carbon nano-immune label chromatography test strip and method for detecting antibiotics in animal hair |
| CN113484519A (en) * | 2021-07-21 | 2021-10-08 | 西北农林科技大学 | Probe, method for detecting zearalenone and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN201489002U (en) * | 2008-11-24 | 2010-05-26 | 上海快灵生物科技有限公司 | High-sensitive food mycotoxin test paper card |
| CN102087284A (en) * | 2010-12-29 | 2011-06-08 | 杭州南开日新生物技术有限公司 | Preparation method of reagent board for rapidly detecting aflatoxin B1 |
-
2013
- 2013-04-03 CN CN201310115725.0A patent/CN103257230B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN201489002U (en) * | 2008-11-24 | 2010-05-26 | 上海快灵生物科技有限公司 | High-sensitive food mycotoxin test paper card |
| CN102087284A (en) * | 2010-12-29 | 2011-06-08 | 杭州南开日新生物技术有限公司 | Preparation method of reagent board for rapidly detecting aflatoxin B1 |
Non-Patent Citations (3)
| Title |
|---|
| RUDOLF KRSKA, ALEXANDRA MOLINELLI: "Rapid test strips for analysis of mycotoxins in food and feed.", 《ANAL BIOANAL CHEM》 * |
| 张甄 等人: "同时检测AFB1和ZEA免疫层析试纸条的研制", 《黑龙江八一农垦大学学报》 * |
| 张甄: "同时检测黄曲霉毒素B_1和玉米赤霉烯酮免疫岑溪试纸条的研制", 《中国优秀硕士论文全文数据库》 * |
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| CN105044346A (en) * | 2015-06-25 | 2015-11-11 | 浙江大学 | Quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and preparation method of quantum dot fluorescence immunochromatography test strip |
| CN105334319A (en) * | 2015-10-21 | 2016-02-17 | 黑龙江省乳品工业技术开发中心 | Test strip for detecting zearalonone in milk |
| WO2018121677A1 (en) * | 2016-12-31 | 2018-07-05 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatography kit for simultaneous detection of mixed pollutant of aflatoxin and carbaryl, preparation method therefor, and application thereof |
| CN106977600A (en) * | 2017-03-07 | 2017-07-25 | 中国农业科学院油料作物研究所 | Purify fumonisin B1, aflatoxin, ochratoxin A, zearalenone immunosorbent and compound affinity column |
| CN106977600B (en) * | 2017-03-07 | 2019-08-13 | 中国农业科学院油料作物研究所 | Purify fumonisin B1, aflatoxin, ochratoxin A, zearalenone immunosorbent and compound affinity column |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN110618274A (en) * | 2019-09-04 | 2019-12-27 | 山东绿都生物科技有限公司 | Chimeric ELISA kit for simultaneously and quantitatively detecting total amount of aflatoxin and zearalenone in edible oil |
| CN110618274B (en) * | 2019-09-04 | 2022-09-20 | 山东绿都生物科技有限公司 | Chimeric ELISA kit for simultaneously and quantitatively detecting total amount of aflatoxin and zearalenone in edible oil |
| CN110806476A (en) * | 2019-11-15 | 2020-02-18 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting sickle knife fungus enol pollution of ribes diacetylenii, preparation method and application thereof |
| CN111007247A (en) * | 2019-11-15 | 2020-04-14 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatographic test strip for synchronously detecting ribes diacetate sickle-knife fungus enol, deoxynivalenol and T-2 toxin |
| CN111007247B (en) * | 2019-11-15 | 2021-09-21 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatographic test strip for synchronously detecting ribes diacetate sickle-knife fungus enol, deoxynivalenol and T-2 toxin |
| CN110806476B (en) * | 2019-11-15 | 2021-10-08 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting sickle knife fungus enol pollution of ribes diacetylenii, preparation method and application thereof |
| CN112305216A (en) * | 2020-10-26 | 2021-02-02 | 杭州电子科技大学 | Carbon nano-immune label chromatography test strip and method for detecting antibiotics in animal hair |
| CN113484519A (en) * | 2021-07-21 | 2021-10-08 | 西北农林科技大学 | Probe, method for detecting zearalenone and application |
| CN113484519B (en) * | 2021-07-21 | 2023-04-28 | 西北农林科技大学 | Probe, method for detecting zearalenone and application |
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