CN103278630B - Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof - Google Patents
Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and a preparation method and an application thereof. The test strip comprises a cardboard, a water absorption pad, a detection pad, a colloidal gold pad and a sample pad are sequentially pasted on one surface of the cardboard from top to bottom, adjacent pads are connected at the connection in an overlapping manner, the detection pad treats a nitrocellulose membrane as a base pad, the nitrocellulose membrane is provided with a transverse quality control line and detection lines, the detection lines are positioned below the quality control line, the number of the detection lines is two, the detection lines are distributed in an interval manner and are coated with an ochratoxin A-bovine serum albumin conjugate and an aflatoxin B1-bovine serum albumin conjugate respectively, and the quality control line is coated with a rabbit anti-mouse polyclonal antibody; and the colloidal gold pad is transversely sprayed with a nano-gold labeled anti-aflatoxin universal monoclonal antibody and a nano-gold labeled anti-zearalenone monoclonal antibody. The immunity chromatography test strip can be used for synchronously detecting the contents of aflatoxin and ochratoxin A in a sample, and has the characteristics of simple operation, rapidness and high sensitivity.
Description
Technical field
The present invention relates to mycotoxin immuno-chromatographic test paper strip, be specifically related to immuno-chromatographic test paper strip, preparation method and the application thereof of a kind of synchronous detection aflatoxin and ochratoxin A composite pollution.
Background technology
Mycotoxin (mycotoxins) is the poisonous secondary metabolite that fungi produces in growth course, and mycotoxin is lower-molecular substance normally, and most mycotoxin all belongs to thermally-stabilised material.Since being found from mycotoxin, the mycotoxin confirmed is nearly more than 300 more than kind, and wherein aflatoxin and ochratoxin are two toxoids of toxicity maximum.Aflatoxin is classified as a class carcinogenic substance by international cancer research institution, and the ochratoxin A in ochratoxin is listed in two class carcinogenic substances.The two is strong toxicity not only, and pollution range is extremely wide, aflatoxin has many reports to the harm of grain and feed, ochratoxin A is also detected a large amount of pollution cereal crops by many countries, has in addition data to show that the two also easily pollutes same cereal crops simultaneously and grain and feed are produced to composite pollution.Aflatoxin can destroy the liver organization of humans and animals strongly, and ochratoxin mainly causes kidney injury, and has the additive effect of toxicity.In view of the damaging effect of mycotoxin and the extensive generation in grain and feed thereof, strict restriction has been carried out to its content in countries in the world.Along with improving constantly of living standard, people are more and more higher to the requirement of Safety of Food Quality.In order to improve China's Safety of Food Quality level, meet greatly people's safe consumption demand, should strengthen the monitoring to mycotoxin in grain, develop detection technique accurately and efficiently, especially Fast Detection Technique, shortens analysis time, improves China's foodsafety.
Existing the detection method of aflatoxin and ochratoxin A is mainly comprised to thin layer chromatography, exact instrument analytic approach and immune analysis method (take enzyme-linked immunosorbent assay and immuno-chromatographic test paper strip as main).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.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 instrument is expensive, the degree of purification of the sample that requirement detects is high, and sample pretreatment process is loaded down with trivial details, length consuming time, experimental situation and testing staff are required high, be difficult to realize fast detecting.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 in recent years, obtained fast development.Immuno-chromatographic test paper strip 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, is widely applied in recent years in qualitative, online, the fast detecting of the minimal residue things such as mycotoxin.Yet the existing colloidal gold immuno-chromatography test paper strip detecting for mycotoxin mostly can only detect a kind of mycotoxin.And the planting patterns of China smallholder decentralized, in agricultural product, mycotoxin incidence is high, and the possibility of same agricultural product mycotoxin composite pollution is large, therefore in the urgent need to fast, synchronously detecting the detection technique of mycotoxin composite pollution, to realize synchronous, the fast monitored to mycotoxin composite pollution in grain and feed.
Summary of the invention
Problem to be solved by this invention is to provide a kind of immuno-chromatographic test paper strip, preparation method and application thereof that can synchronously detect aflatoxin and ochratoxin A composite pollution.This immuno-chromatographic test paper strip can be used for the synchronous detection of aflatoxin and ochratoxin A content in sample, has simple to operate, quick, highly sensitive feature.
The present invention solves the problems of the technologies described above adopted technical scheme to be:
The synchronous immuno-chromatographic test paper strip (seeing Fig. 1 and Fig. 2) that detects aflatoxin and ochratoxin A 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, on described two detection lines, be coated with respectively ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) and aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA), described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, described gold mark pad transverse jet scribbles the anti-ochratoxin A 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 1H2 secretion that anti-ochratoxin A monoclonal antibody is CCTCC NO.C201329 by deposit number produces, this hybridoma cell strain 1H2 has been preserved in Chinese Typical Representative culture collection center (CCTCC) on March 7th, 2013, preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO.C201329.
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, and on the detection line of close nature controlling line and nitrocellulose filter, the spacing on edge is 15～20mm, and described is 5～7mm near the detection line of nature controlling line and the spacing of nature controlling line.
Press such scheme, the package amount that described detecting pad is coated with every centimetre of needed ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) on the detection line of ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) 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-ochratoxin A monoclonal antibody of required nano gold mark is 100～200ng.
The preparation method of the immuno-chromatographic test paper strip of synchronous detection aflatoxin as above and ochratoxin A composite pollution, comprises 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 the conjugate of ochratoxin A-bovine serum albumin(BSA) (OTA-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, be dried 30～60 minutes; On the detection line of the conjugate of described coated ochratoxin A-bovine serum albumin(BSA) (OTA-BSA), the package amount of the conjugate (OTA-BSA) of every centimetre of needed ochratoxin A-bovine serum albumin(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 coating buffer of 0.2～0.4mg/mL with coated damping fluid, in the position of the detection line 5～7mm apart near nature controlling line, by a spray mode, it is laterally 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, is dried 1～2 hour;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and soak, take out, under 37～40 ℃ of conditions, be dried 6～10 hours, obtain sample pad, then put room temperature preservation in exsiccator;
(4) preparation of gold mark pad
Glass fibre membrane is put into confining liquid to soak, take out, under 37～40 ℃ of conditions, be dried 6～10 hours, by a spray mode, on dry glass fibre membrane, laterally spray the mixed solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of nano gold mark and the anti-ochratoxin A monoclonal antibody solution of nano gold mark, 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-ochratoxin A monoclonal antibody of required nano gold mark is 100～200ng, 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 1H2 secretion that anti-ochratoxin A monoclonal antibody is CCTCC NO.C201329 by deposit number produces,
(5) assembling of test strips
In the one side of cardboard, paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, overlapping length is 1～3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution.
Press such scheme, in the described every 10mL of 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.
Press such scheme, in the every 100mL of confining liquid using in described step (3) and step (4), contain: oralbumin 1～2g, sucrose 2～5g, 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, the general monoclonal antibody solution of 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 5.0mL concentrate, put 4 ℃ of refrigerators standby;
The anti-ochratoxin A monoclonal antibody 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the anti-ochratoxin A monoclonal antibody aqueous solution that slowly adds 1.5mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 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: 2.0g PEG-400, and 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 ochratoxin A composite pollution, method is as follows: take levigate testing sample, adding volumetric concentration is 60～80% methanol aqueous solution, mix, under 50～60 ℃ of water-baths, ultrasonic extraction is 5～10 minutes, standing 5～10 minutes, by supernatant liquor, it is extract dilute with water, the final volume concentration that makes methyl alcohol in dilution is 20～30%, obtain testing sample solution, getting this testing sample solution of 80-150 μ L detects as detecting liquid and dropwise join in the sample pad of immuno-chromatographic test paper strip of synchronous detection aflatoxin and ochratoxin A composite pollution again, it is as test strip, separately get the consistent methanol aqueous solution of isopyknic methanol concentration as negative controls, dropwise add in the sample pad of another immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar: when in test strip, the detection line color of conjugate (OTA-BSA) of coated ochratoxin A-bovine serum albumin(BSA) and the color of corresponding detection line on control stripes bar approach, show in testing sample solution that ochratoxin A content is lower than 0.5ng/mL, during than corresponding detection line of light color, show in testing sample solution that ochratoxin A content is equal to or higher than 0.5ng/mL and lower than 2ng/mL, while not developing the color, show that in testing sample solution, the content of ochratoxin A is equal to or higher than 2ng/mL,
When the detection line color of conjugate (AFB1-BSA) of coated aflatoxin B1-bovine serum albumin(BSA) and the color of corresponding detection line on control stripes bar approach in 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 ochratoxin A in testing sample.
Immuno-chromatographic test paper strip provided by the invention is in the synchronous principle of work detecting in aflatoxin and ochratoxin A composite pollution application: when testing sample solution joins in the sample pad of test strips lower end, testing sample solution moves to 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-ochratoxin A monoclonal antibody of nano gold mark are dissolved.While containing aflatoxin in sample, aflatoxin by 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 is by limited antigen binding site on the aspergillus flavus resisting toxin general purpose single clonal antibody with aflatoxin competition combining nano gold mark, 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 forming on detection line is more shallow, while containing ochratoxin A in sample, ochratoxin A by with gold mark pad on nano gold mark anti-ochratoxin A monoclonal antibody in conjunction with and together upwards swimming, when its arrival is fixed wtih the detection line I of ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) antigen, antigen is by limited antigen binding site in the anti-ochratoxin A monoclonal antibody with ochratoxin A competition combining nano gold mark, in sample, ochratoxin A content is higher, antigen on detection line can in conjunction with the anti-ochratoxin A monoclonal antibody of nano gold mark will be fewer, the colour developing band color forming on detection line is more shallow.When the corresponding antibody 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, and the bond of mycotoxin moves to nature controlling line by continuing and the anti-mouse polyclonal antibody of the rabbit on nature controlling line is combined and is developed the color by enrichment for the antibody of antimycotic toxin of the nano gold mark that the antigen on not tested survey line is intercepted and captured or the antibody of the antimycotic toxin of nano gold mark.Accordingly, respectively the detection line of conjugate and the detection line of coated ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) of coated aflatoxin B1-bovine serum albumin(BSA) in test strip are developed the color and contrasted with corresponding detection line color on control stripes bar, can obtain the composite pollution situation of aflatoxin and these two kinds of mycotoxins of ochratoxin A in sample.
Beneficial effect of the present invention:
(1) one step, detect aflatoxin and ochratoxin A composite pollution simultaneously.Immuno-chromatographic test paper strip provided by the invention can be realized synchronous, the fast detecting to aflatoxin and these two kinds of mycotoxins of ochratoxin A in a test strips, the antibody using is monoclonal antibody, specificity is good, highly sensitive, noiseless between the detection of each mycotoxin, simple, quick.
(3) highly sensitive.Immuno-chromatographic test paper strip provided by the invention is limited to 0.25ng/mL to detecting the lowest detection of aflatoxin in solution, lowest detection to ochratoxin A is limited to 0.5ng/mL, and this detectability can meet the limit the quantity of requirement of European Union to these two kinds of mycotoxins in food.
(2) simple to operate.Sample pre-treatments only need to add methanol-water extract in sample ultrasonic extraction 5～10 minutes, and then standing 5～10 minutes, to get supernatant dilution and can detect, whole sample pretreatment process is simple, fast.
Accompanying drawing explanation
Fig. 1 is the front elevation of the immuno-chromatographic test paper strip of synchronous detection aflatoxin of the present invention and ochratoxin A composite pollution;
Fig. 2 is the side view of the immuno-chromatographic test paper strip of synchronous detection aflatoxin of the present invention and ochratoxin A composite pollution;
Fig. 3 be embodiment 2 result process decision chart;
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-ochratoxin A 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, preparation method is: the BALB/c mouse that the hybridoma cell strain 1C11 injection that is CCTCC NO.C201018 by deposit number was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt 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, under stirring, slowly add caprylic acid, 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 obtaining 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, with the sodium hydroxide solution of 2mol/L, regulate the pH value to 7.4 of this mixed liquor, 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, gained precipitation is resuspended with the 0.01mol/L phosphate buffer of former ascites volume 1/10, pack bag filter into, pure water is dialysed, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect freeze-dried powder, obtain the aspergillus flavus resisting toxin monoclone antibody that purifying is good, antibody is placed in to-20 ℃ of refrigerators standby,
Described acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to 100mL gained; The phosphate buffer of described 0.1mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g, adds water and is settled to 100mL gained.
B. the hybridoma cell strain 1H2 that anti-ochratoxin A monoclonal antibody is CCTCC NO.C201329 by deposit number produces, its preparation method is: the BALB/c mouse that hybridoma cell strain 1H2 injection was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt 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, under stirring, slowly add caprylic acid, 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 obtaining 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, with the sodium hydroxide solution of 2mol/L, regulate the pH value to 7.4 of this mixed liquor, 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 gained precipitation with former ascites volume 1/10, pH value is that 7.4 phosphate buffer is resuspended, pack bag filter into, pure water is dialysed, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect freeze-dried powder, obtain the anti-ochratoxin A monoclonal antibody that purifying is good, antibody is placed in to-20 ℃ of refrigerators standby,
Described acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to 100mL gained; The phosphate buffer of described 0.1mol/L is 8g sodium chloride, 2.9g disodium hydrogen phosphate, and 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g, adds water constant volume to 100mL gained; The phosphate buffer of described 0.01mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g, adds water and is settled to 100mL gained.
The hypotype of identifying the anti-ochratoxin A monoclonal antibody of hybridoma cell strain 1H2 secretion with commercially available hypotype identification kit is IgG1.
With the BALB/c mouse ascites antibody that the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method records injection hybridoma cell strain 1H2, tire and can reach 7.2 * 10
5, mouse ascites antibody dilution 7.2 * 10
5times time measured in solution result positive.By conventional indirect competitive ELISA method, identify that it is 52pg/mL to the sensitivity of ochratoxin A, with ochratoxin B, aflatoxin B1, B2, G1, G2, vomitoxin, zearalenone, the cross reacting rate of fumonisin is all less than 0.1%.
The screening of hybridoma cell strain 1H2:
(1) animal immune
Buy 6 of BALB/c mouse in 6 week age, the ochratoxin A comlete antigen OTA-BSA that immunity is commercially available.Immunity is by after ochratoxin A 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.After being immune to for the second time 4 weeks, carry out, adopt freund 's incomplete adjuvant and the emulsification of isopyknic ochratoxin A comlete antigen, in mouse peritoneal, inject.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 70 μ g.Latter 8～10 days of 3 times each immunity, tail vein blood, separation of serum, adopts 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 indirect competitive ELISA method, selection is tired, sensitivity all relatively high mouse corresponding to serum carry out last booster immunization, immunizing dose is above 2 times.
Ochratoxin A comlete antigen OTA-BSA is purchased from Sigma-Aldrich company.
(2) Fusion of Cells
In last booster immunization after 3 days, adopt 50%(percent by weight) polyglycol be that PEG(molecular weight is 1450) make fusion agent, carry out according to a conventional method Fusion of Cells, concrete steps: kill immune mouse under aseptic condition, separating Morr. cell, with mouse source myeloma cell SP2/0 with the number of 5 ︰ 1 than mixing, with RPMI-1640 basic culture solution, wash cell mixing, with 50%PEG, merge, merge 1 minute, then slowly add 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 having hanged is joined in 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) dual anti-(the 10000 every ml penicillins of unit and every milliliter of streptomysin 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 1%(mass percent) the cell complete medium of methylcellulose; RPMI-1640 basic culture solution, HEPES, dual 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, with micropipettor, clone is drawn from this nutrient culture media, 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 ELISA method to there being the culture hole of Growth of Hybridoma Cell to screen, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out anti-ochratoxin A and the positive hole of not anti-carrier protein BSA; The positive hole that second step adopts indirect competitive ELISA method to filter out the first step is detected, former as competition with ochratoxin A, 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 detect, so repeat after subclone 2-3 time acquisition hybridoma cell strain 1H2.
Hybridoma cell strain 1H2 antibody variable region sequencing
(1) extract total RNA: adopt the total RNA extraction reagent box of Tian Gen company and extract to specifications total RNA that can produce hybridoma cell strain 1H2;
(2) synthetic cDNA: total RNA that the step 1 of take obtains is template, oligo (dT)
15for primer, according to SuperScript
tM-2 II reverse transcriptase instructionss carry 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.PCR program is: 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.PCR product is through 1%(percent by weight) agarose gel electrophoresis separation after, with kit, purify and reclaim DNA fragmentation, be connected in carrier pMD18-T, transform bacillus coli DH 5 alpha competent cell, picking positive colony, delivers to Sani bio tech ltd, Shanghai and checks order.Wherein the sequence of primer is respectively: variable region of heavy chain primer is that (22mer) (32mer) wherein S, M, R and W are 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 obtaining: the long 353bp of variable region of heavy chain coding gene sequence, sequence is as shown in SEQ ID NO:1, according to obtained gene order, derive the coded variable region of heavy chain of this gene order and be comprised of 117 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 ID NO:4.
The synchronous preparation method who detects the immuno-chromatographic test paper strip of aflatoxin and ochratoxin A composite pollution, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out to growth 16mm, and the specification of wide 4mm, obtains adsorptive pads;
(2) preparation of detecting pad
Ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) is mixed with to the coating buffer of 0.4mg/mL with coated damping fluid, in on nitrocellulose filter along the position of 15mm, by a spray mode, be coated on nitrocellulose filter and obtain detection line I, in every centimetre of detection line I, the package amount of required ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) is 150ng; Aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is mixed with to the solution of 0.25mg/mL with coated damping fluid, in the position apart from detection line I 2mm, by a some spray mode, be coated in and on nitrocellulose filter, obtained detection line II, in every centimetre of detection line II, the package amount of required aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 100ng, then under 37 ℃ of conditions, is dried 30 minutes;
The long 22mm of described nitrocellulose filter, wide 4mm;
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, in the position apart from detection line I 6mm, by a spray mode, it is laterally 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 80ng, then under 37 ℃ of conditions, is dried 1 hour;
Described coated damping fluid is: 0.1g bovine serum albumin(BSA), and 0.08g sodium chloride, 0.029g disodium hydrogen phosphate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate, adds water constant volume to 10mL gained;
(3) preparation of sample pad
Glass fibre membrane is cut out to growth 12mm, and the specification of wide 4mm, puts into confining liquid and soaks, and takes out, and under 37 ℃ of conditions, is dried 8 hours, obtains sample pad, then puts room temperature preservation in exsiccator;
Described confining liquid is: by 1g oralbumin, and 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, adds water and is settled to 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 the described confining liquid of step (3) soaks, take out, under 37 ℃ of conditions, be dried 8 hours, on dry glass fibre membrane, by a spray mode, on dry glass fibre membrane, laterally spray the mixed solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of nano gold mark and the anti-ochratoxin A monoclonal antibody of nano gold mark, 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 125ng, the consumption of the anti-ochratoxin A monoclonal antibody of required nano gold mark is 150ng, then vacuum freeze drying is 2 hours, put room temperature preservation in exsiccator,
The general monoclonal antibody solution of 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 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-ochratoxin A monoclonal antibody 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the anti-ochratoxin A monoclonal antibody aqueous solution that slowly adds 1.5mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 5.0mL concentrate, put 4 ℃ of refrigerators standby, and wherein the mass concentration of the anti-ochratoxin A monoclonal antibody solution of nano gold mark is 0.03mg/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: 2.0g PEG-400, and 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
In the one side of cardboard, paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, overlapping length is 1～3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution, sees Fig. 1 and Fig. 2.
The application of the immuno-chromatographic test paper strip of above-mentioned synchronous detection aflatoxin and ochratoxin A composite pollution in peanut sample detects:
Take levigate 1#, 2# and 3# peanut sample to be measured, adding volumetric concentration is 70% methanol aqueous solution, 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 testing sample solution, get again 100 μ L testing sample solutions as detect liquid dropwise add a synchronous immuno-chromatographic test paper strip that detects aflatoxin and ochratoxin A composite pollution sample pad, 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 immuno-chromatographic test paper strip that detects aflatoxin and ochratoxin A composite pollution sample pad, 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, detection line I color is more shallow than contrast ELISA test strip line I, detection line II does not develop the color, see Fig. 3-1, judge thus: in 1# testing sample solution, the content of ochratoxin A is equal to or higher than 0.5ng/mL and lower than 2ng/mL, the content of aflatoxin is equal to or higher than 1ng/mL; The content can obtain ochratoxin A in 2# testing sample through converting is equal to or higher than 6ng/g and lower than 24ng/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 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. 3-2, judge thus: in 2# testing sample solution, the content of ochratoxin A is lower than 0.5ng/mL, and the content of aflatoxin is equal to or higher than 0.25ng/mL and lower than 1ng/mL; The content that can obtain ochratoxin A in 2# testing sample through converting is lower than 6ng/g; The content of aflatoxin is equal to or higher than 3ng/g, and lower than 12ng/g.
The nature controlling line of 3# testing sample test strip demonstrates red stripes, and detection line I and detection line II all do not develop the color, and see Fig. 3-3, judges thus: in 3# testing sample solution, the content of ochratoxin A is equal to or higher than 2ng/mL; The content of aflatoxin is equal to or higher than 1ng/mL; The content that can obtain ochratoxin A in 3# testing sample through converting is equal to or higher than 24ng/g; The content of aflatoxin is equal to or higher than 12ng/g.
Embodiment 3:
The synchronous preparation method who detects the immuno-chromatographic test paper strip of aflatoxin and ochratoxin A composite pollution, step is as follows:
(1) preparation of adsorptive pads
Thieving paper is cut out to growth 18mm, and the specification of wide 3mm, obtains adsorptive pads;
(2) preparation of detecting pad
Ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-BSA) is mixed with to the coating buffer of 0.4mg/mL with coated damping fluid, in on nitrocellulose filter along the position of 20mm, by a spray mode, be coated on nitrocellulose filter and obtain detection line I, in every centimetre of detection line I, the package amount of required ochratoxin A-bovine serum albumin(BSA) conjugate (OTA-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, in the position apart from detection line I 4mm, by a some spray mode, be coated in and on nitrocellulose filter, obtained detection line II, in every centimetre of detection line II, the package amount of required aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) is 300ng, then under 40 ℃ of conditions, is dried 30 minutes;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with to the coating buffer of 0.5mg/mL with coated damping fluid, in the position apart from detection line I 5mm, by a spray mode, it is laterally 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 200ng, then under 40 ℃ of conditions, is dried 1 hour;
Described coated damping fluid is: 0.2g bovine serum albumin(BSA), and 0.08g sodium chloride, 0.029g disodium hydrogen phosphate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate, adds water constant volume to 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, puts into confining liquid and soaks, and takes out, and under 40 ℃ of conditions, is dried 6 hours, obtains sample pad, then puts room temperature preservation in exsiccator.
Described confining liquid is: by 2g oralbumin, and 4g sucrose, 0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, adds water and is settled to 100mL gained;
(4) preparation of gold mark pad
Glass fibre membrane is cut out to growth 12mm, the specification of wide 3mm, the confining liquid of putting into step (3) soaks, take out, under 37 ℃ of conditions, be dried 8 hours, on dry glass fibre membrane, by a spray mode, on dry glass fibre membrane, laterally spray the mixed solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of nano gold mark and the anti-ochratoxin A monoclonal antibody of nano gold mark, 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-ochratoxin A monoclonal antibody of required nano gold mark is 100ng, then vacuum freeze drying is 2 hours, put room temperature preservation in exsiccator,
The preparation method that the general monoclonal antibody solution of aspergillus flavus resisting toxin of described nano gold mark and the anti-ochratoxin A monoclonal antibody solution of nano gold mark are is as embodiment 2, and difference is; In its nano-Au solution using, the particle diameter of nm of gold is 20nm;
(5) assembling of test strips
In the one side of cardboard, paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, overlapping length is 3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution.
The application of the immuno-chromatographic test paper strip of above-mentioned synchronous detection aflatoxin and ochratoxin A composite pollution in peanut sample detects
Take levigate peanut sample to be measured, adding volumetric concentration is 60% methanol aqueous solution, 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 testing sample solution, get again 100 μ L testing sample solutions as detect liquid dropwise add a synchronous immuno-chromatographic test paper strip that detects aflatoxin and ochratoxin A composite pollution sample pad, 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 immuno-chromatographic test paper strip that detects aflatoxin and ochratoxin A composite pollution sample pad, 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, detection line I and detection line II all do not develop the color, and judge thus: in testing sample solution, the content of ochratoxin A is equal to or higher than 2ng/mL; In testing sample solution, the content of aflatoxin is equal to or higher than 1ng/mL; Through converting, can obtain the content of ochratoxin A in testing sample and be equal to or higher than 24ng/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 ochratoxin A 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, on described two detection lines, be coated with respectively ochratoxin A-bovine serum albumin(BSA) conjugate and aflatoxin B1-bovine serum albumin(BSA) conjugate, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, described gold mark pad transverse jet scribbles the anti-ochratoxin A 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 1H2 secretion that anti-ochratoxin A monoclonal antibody is CCTCC NO.C201329 by deposit number produces.
2. the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 1 and ochratoxin A 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 ochratoxin A 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 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 ochratoxin A composite pollution, is characterized in that: the package amount that described detecting pad is coated with every centimetre of needed ochratoxin A-bovine serum albumin(BSA) conjugate on the detection line of ochratoxin A-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 ochratoxin A 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-ochratoxin A monoclonal antibody of required nano gold mark is 100～200ng.
6. synchronously detect the preparation method of the immuno-chromatographic test paper strip of aflatoxin and ochratoxin A composite pollution, it is characterized in that: it comprises the following steps:
(1) 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 the conjugate of ochratoxin A-bovine serum albumin(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, be dried 30～60 minutes; On the detection line of the conjugate of described coated ochratoxin A-bovine serum albumin(BSA), the package amount of the conjugate of every centimetre of needed ochratoxin A-bovine serum albumin(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 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 coating buffer of 0.2～0.4mg/mL with coated damping fluid, in the position of the detection line 5～7mm apart near nature controlling line, by a spray mode, it is laterally 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, is dried 1～2 hour;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and soak, take out, under 37～40 ℃ of conditions, be dried 6～10 hours, obtain sample pad, then put room temperature preservation in exsiccator;
(4) preparation of gold mark pad
Glass fibre membrane is put into confining liquid to soak, take out, under 37～40 ℃ of conditions, be dried 6～10 hours, by a spray mode, on dry glass fibre membrane, laterally spray the mixed solution of the general monoclonal antibody solution of aspergillus flavus resisting toxin of nano gold mark and the anti-ochratoxin A monoclonal antibody solution of nano gold mark, 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-ochratoxin A monoclonal antibody of required nano gold mark is 100～200ng, 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 1H2 secretion that anti-ochratoxin A monoclonal antibody is CCTCC NO.C201329 by deposit number produces,
(5) assembling of test strips
In the one side of cardboard, paste successively from top to bottom adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, overlapping length is 1～3mm, obtains the immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution.
7. the preparation method of the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 6 and ochratoxin A composite pollution, it is characterized in that: in the described every 10mL of 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 of the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 6 and ochratoxin A composite pollution, it is characterized in that: in the every 100mL of confining liquid using in described step (3) and step (4), contain: oralbumin 1～2g, sucrose 2～5g, sodium azide 0.02～0.05g, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
9. the preparation method of the immuno-chromatographic test paper strip of synchronous detection aflatoxin according to claim 6 and ochratoxin A composite pollution, it is characterized in that: the general monoclonal antibody solution of 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 5.0mL concentrate, put 4 ℃ of refrigerators standby;
The anti-ochratoxin A monoclonal antibody 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%, with 0.4mL0.1mol/L wet chemical, regulate pH value, the anti-ochratoxin A monoclonal antibody aqueous solution that slowly adds 1.5mL0.1mg/mL under the state stirring, continues 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, gets supernatant, abandons precipitation; By the centrifugal 30min of supernatant 12000r/min, abandoning supernatant, adds the washing of 40.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 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: 2.0g PEG-400, and 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 synchronous detection aflatoxin described in any one in claim 1-5 and ochratoxin A composite pollution, it is characterized in that: its application process is: take levigate testing sample, adding volumetric concentration is 60～80% methanol aqueous solution, mix, under 50～60 ℃ of water-baths, ultrasonic extraction is 5～10 minutes, standing 5～10 minutes, by supernatant liquor, it is extract dilute with water, the final volume concentration that makes methyl alcohol in dilution is 20～30%, obtain testing sample solution, getting this testing sample solution of 80-150 μ L detects as detecting liquid and dropwise join in the sample pad of immuno-chromatographic test paper strip of synchronous detection aflatoxin and ochratoxin A composite pollution again, it is as test strip, separately get the consistent methanol aqueous solution of isopyknic methanol concentration as negative controls, dropwise add in the sample pad of another immuno-chromatographic test paper strip that synchronously detects aflatoxin and ochratoxin A composite pollution, it is test strips in contrast, contrast after 15-20 minute develops the color test strip and control stripes bar:
When the detection line color of conjugate of coated ochratoxin A-bovine serum albumin(BSA) and the color of corresponding detection line on control stripes bar approach in test strip, show in testing sample solution that ochratoxin A content is lower than 0.5ng/mL; During than corresponding detection line of light color, show in testing sample solution that ochratoxin A content is equal to or higher than 0.5ng/mL and lower than 2ng/mL; While not developing the color, show that in testing sample solution, the content of ochratoxin A is equal to or higher than 2ng/mL;
When the detection line color of conjugate of coated aflatoxin B1-bovine serum albumin(BSA) and the color of corresponding detection line on control stripes bar approach in 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 ochratoxin A in testing sample.
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| CN103728452B (en) * | 2014-01-23 | 2015-06-24 | 湖南大学 | Colloidal gold immunoassay kit for detecting aflatoxin B1 and preparation method of kit |
| CN104459062A (en) * | 2014-12-08 | 2015-03-25 | 北京市理化分析测试中心 | Magnetic immunochromatographic kit for detecting ochratoxin A and preparation method of magnetic immunochromatographic kit |
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| CN106918704B (en) * | 2016-12-31 | 2019-01-04 | 中国农业科学院油料作物研究所 | Synchronous detection aflatoxin and the time-resolved fluoroimmunoassay of carbaryl composite pollution chromatograph kit, preparation method and application |
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| 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 |
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