CN1168986C - Aflatoxin fast detecting apparatus and its making method - Google Patents
Aflatoxin fast detecting apparatus and its making method Download PDFInfo
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- CN1168986C CN1168986C CNB021019517A CN02101951A CN1168986C CN 1168986 C CN1168986 C CN 1168986C CN B021019517 A CNB021019517 A CN B021019517A CN 02101951 A CN02101951 A CN 02101951A CN 1168986 C CN1168986 C CN 1168986C
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Abstract
The present invention relates to a device for rapidly detecting aflatoxins and a manufacturing method thereof. The device is used for detecting harmful metabolites generated by fungi, particularly for detecting aflatoxins. Aflatoxins in a sample are rapidly detected at low cost under the conditions of not needing special devices nor depending professional staff. The device has the main technical scheme that filter paper, a polyester cellulose pad, a cellulose nitrate film and filter paper are orderly attached to a long plastic bottom board from one end to the other end, and one layer of colloidal gold-monoclonal antibodies with the spray quantity of 50 to 80 OD/milliliter is sprayed on the polyester cellulose pad; aflatoxin-bovine serum albumin complexes and sheep anti white mouse immune globulin G1 antibodies which have the concentration of 1 to 2 milligrams/milliliter and the spray quantity of 0.7 to 1.5 microliters/centimeter are transversally sprayed on the cellulose nitrate film at intervals.
Description
Technical field
The present invention relates to detection, particularly to the fast detecting of aflatoxin to metabolin that fungi produces.
Background technology
As everyone knows, aflatoxin is the extremely strong material of a kind of toxicity, is again strong carcinogen, and its generation and pollution are to be undertaken by feed, cereal and food mostly.Harm to people, animal is quite big.Therefore, cause that the scientific worker payes attention to greatly, and take much aflatoxin to be suppressed, prevent its diffusion, avoid measures such as people, animal poisoning.And be an important step of described measure to its qualitative and detection by quantitative that in feed and cereal, exists.Known method to the aflatoxin detection, mainly contain thin layer chromatography (referring to 1998 01 phases of Chinese sanitary inspection magazine), euzymelinked immunosorbent assay (ELISA) (referring to Indian Veterinary Journal (1997,74 phase)) and liquid phase chromatography (referring to the magazine chromatogram, 04 phase of nineteen ninety-five).Thin layer chromatography is to adopt the silica G thin plate to carry out the toxin chromatography, under 365 nano-ultraviolet lights, observe the blue fluorescence of aflatoxin generation and (see GB/T5009 with definite existing of toxin with the mobility ratio of normaltoxin, 22-1996), euzymelinked immunosorbent assay (ELISA) is to adopt aflatoxin-protein complexes bag by elisa plate, then the antibody of sample with the identification aflatoxin is mixed, in the aperture of elisa plate, carry out immune response, determine the concentration of aflatoxin in the sample by colorimetric, the accuracy of detection of this method is 10ppb, and the precision of the aflatoxin of liquid chromatography for measuring is similar to euzymelinked immunosorbent assay (ELISA).All there is following shortcoming in above-mentioned each method: the running program complexity, can only carry out in the laboratory; Need the professional to operate, with special instrument and equipment, detection time long (generally needing 3-10 days), required expense height, its expense is at 200-1000 yuan/each sample.
Summary of the invention
The object of the invention is to provide a kind of fast detecting examination of aflatoxin device and manufacture method thereof, it can be operated at the scene, and can participate in by non-specialized-technical personnel, do not need any instrument and equipment, with lower expense, just can detect existence and the content that contains aflatoxin in the sample in the short period of time.
Purpose of the present invention can reach by following measure:
The feature of this aflatoxin fast detecting apparatus is: from one end to the other side overlapping mutually successively on the rectangular base plate of plastics (5) attaches filter paper (1-1), the plain pad of polyester fiber (2), nitrocellulose filter (3) and filter paper (1-2), the attached one deck of spray absorbance under 540 nano wave lengths is the collaurum-monoclonal anti nanocrystal composition (21) of the concentration of 0.5-0.8 on the plain pad of described polyester fiber, monoclonal antibody is wherein produced by hybridoma and selectivity identification aflatoxin, and this hybridoma is via aflatoxin AFB
1-BSA albumen composition immunity small white mouse, obtain by the method for Davis again, purified monoclonal antibody is coated on and makes collaurum-monoclonal anti nanocrystal composition on the collaurum of being made by gold chloride, its particle size is the 18-20 nanometer, be peony, absorbance is the concentration of 0.5-0.8 under 540 nano wave lengths; On described nitrocellulose filter, laterally spray attached following two kinds of materials separately: concentration be the spray cloth amount of 1～2 mg/ml be 0.7～1.5 microlitre/centimetre aflatoxin-bovine serum albumin(BSA) complex (31), wherein molecular weight is that 66200 daltonian each bovine serum albumin molecule combine with 11-14 aflatoxin molecule; Concentration is the 1-2 mg/ml, spray cloth amount be 0.7～1.5 microlitre/centimetre goat-anti small white mouse IgG
1Antibody (32), it is by injection small white mouse immunoglobulin (Ig) in the goat body, get its serum that contains antibody and purified after make, its molecular weight is 80.000 dalton, on polyacrylamide gel electrophoresis, present two bands, article one, be that molecular weight is 55000 dalton, another is that molecular weight is 25.000 dalton.
Because monoclonal antibody is to be produced by the filial generation of a hybridoma, so the monoclonal antibody uniform quality that produces, and monoclonal antibody recognition objective molecule or structure is very capable, the selectivity combination that it has, homogeneity antibody, the also characteristics of energy long term storage can be mass-produced.Make the suitability for industrialized production of its application and corresponding product become possibility.If splash into a certain amount of sample at the filter paper place of detecting the close collaurum-monoclonal antibody of test paper, when not having aflatoxin in the mixed liquor, then mixed liquor will be moved to aflatoxin-bovine serum albumin(BSA) complex zone and just combine the compound of generation peach collaurum-monoclonal antibody---aflatoxin---bovine serum albumin(BSA) complex at 5-10 minute with its its with collaurum-monoclonal anti nanocrystal composition; When having aflatoxin in the mixed liquor, then it also will be at 5-10 minute just produces collaurum---monoclonal antibody---aflatoxin compound with collaurum-monoclonal anti nanocrystal composition, this compound can not with the AFB that is fixed on the nitrocellulose filter
1-BSA combination, therefore this zone is colourless, when the quantity of the aflatoxin that contains in the mixed liquor is less than the quantity of collaurum-monoclonal anti nanocrystal composition, its remaining collaurum-monoclonal anti nanocrystal composition will combine the compound that generates peach collaurum-monoclonal antibody-aflatoxin-bovine serum albumin(BSA) complex with aflatoxin-bovine serum albumin(BSA) complex, just because the quantity of combination is few shown pink shallow.So work as and standard aflatoxin sample, for example concentration is 0,2,4,6,8 ... 20/ milliliter when measuring simultaneously, can estimate in the pink depth that aflatoxin-bovine serum albumin(BSA) complex place manifests and to contain what of aflatoxin amount in the sample mix liquid.Because the goat-anti small white mouse IgG on nitrocellulose filter
1Antibody run into come from the monoclonal antibody that small white mouse is attached on the collaurum also can the pulverize redness, therefore, this peach formation can be the validity that the tester points out this pick-up unit, promptly has pink to be produced as Validity Test, does not have that pink colo(u)r streak is produced as invalid test or product is out of date.
The manufacture method of aflatoxin fast detecting apparatus:
(A) attach filter paper (1-1) at the right-hand member that scribbles the base plate of adhesive sticker (5), with transferring in absorbance under 540 nano wave lengths is that collaurum-monoclonal anti nanocrystal composition (21) spray of the concentration of 0.5-0.8 is attached on the polyester fiber pad (2), the polyester fiber pad is attached on the base plate (5), and with the overlapping 1-2 millimeter of filter paper (1-1) of right-hand member; With concentration is the aflatoxin-bovine serum albumin(BSA) complex (31) and the goat-anti small white mouse IgG of 1-2 mg/ml
1Antibody (32) with the 0.7-1.5 microlitre/centimetre spray cloth amount, the spray of the alternate 8-10 of being divided into mm distance is attached on the nitrocellulose filter (3), it is attached on the base plate (5), and with the overlapping 1-2 millimeter of polyester fiber pad (2) of right-hand member; Filter paper (1-2) is attached on the base plate (5), and with the overlapping 1-2 millimeter of nitrocellulose filter (3) of right-hand member; The base plate (5) that posts above-mentioned substance is cut into the rectangular of 4-5 millimeter;
(B) preparation of described collaurum-monoclonal antibody (21) solution: the method for pressing Davis is with aflatoxin-bovine serum albumin(BSA) complex AFB
1-BSA is dissolved in the phosphoric acid physiological saline (PBS) and Fu Shi Freund's complete adjuvant with volume, get 150 micrograms, divide and be injected in the female small white mouse body for three times, per injection 50 micrograms, in two weeks of space, 3～5 days after the injection are got the disconnected neck of the high small white mouse of tiring and are put to death and get spleen cell and NS-1 myeloma cell's fusion for the third time; Put it into and contain 1.25% methylcellulose, in 37 ℃ incubator, cultivate in the semisolid culturemedium of 25% calf serum and HAT nutrient culture media, take out after 7-10 days and put into the RPMI1640 nutrient culture media that contains 10% calf serum HT nutrient culture media and antibiotic again and cultivate; Take out after three days, and get 10 microlitres, in using AFB
1Carry out enzyme linked immunoassay ELISA in 96 orifice plates of-BSA bag quilt, determine to produce the hybridoma of aspergillus flavus resisting toxin antibody, the hybridoma of the generation aspergillus flavus resisting toxin obtained was cultivated 3-5 days in the PRMI1640 nutrient culture media, when treating that antibody concentration reaches the 5-10 mg/ml, sodium chloride concentration in the above-mentioned nutrient solution is transferred to 3.3 moles, and add 1 mole of sodium borate of 1/10 volume, above-mentioned solution is joined in albumin A-SepharoseCL4B post, twice each sodium chloride of 3 moles with 10 times of volumes, the sodium borate flushing chromatographic column of 50 mMs is used 100 mMs, pH value is the albumen in 3.0 the glycine solution wash-out post; Adding 1 mole of 50 microlitres, pH value in the collection tube is 8.0Tris-HCL, collects eluent with it then, and the monoclonal antibody of collection is used for collaurum-MONOCLONAL ANTIBODIES SPECIFIC FOR after dialysis; Get 0.1 gram sodium chloraurate and be dissolved in 1000 milliliters the deionized water, heating is boiled to 60 ℃, put into 50 milliliters of 1% sodium citrate solutions of newly joining, wherein contain citric acid 0.2%, contain tannic acid 0.2%, be heated to then 95 ℃ 5 minutes, formed colloid gold particle diameter is the 18-20 nanometer, monoclonal antibody after the dialysis is centrifugal 30 minutes through 36000 rev/mins, get its supernatant through 0.2 micron membrane filtration, join in the colloidal gold solution after its concentration transferred to 0.5 mg/ml, make its ultimate density reach the 5-10 mcg/ml, the polyvinyl alcohol (PVA) (PEG) that adds 1 milliliter of 1% concentration after 5 minutes in per 100 milliliters of above-mentioned solution to be stoping nonspecific agglutination, with 10000 rev/mins centrifugal 1 hour, remove supernatant, centrifugal 1 hour with 10000 rev/mins again, remove the not protein of absorption, the collaurum of bag quilt is diluted to absorbance is the concentration of 0.5-0.8 under 540 nano wave lengths, go bacterium with 0.2 micron membrane filtration;
(C) the protein complex AFB of described aflatoxin-cow's serum
1The preparation of-BSA solution: dilute aflatoxin-bovine serum albumin(BSA) complex to the 1.2-1.5 mg/ml with phosphoric acid normal saline buffer solution PBS;
(D) described goat-anti small white mouse IgG
1The preparation of solution: the small white mouse immunoglobulin (Ig) is diluted to 10 mg/ml, adds isopyknic Fu Shi Freund's complete adjuvant again; From carry out through animal feeding room goat that adaptability raises get 3～5 milliliters of immunity on one's body before blood sample make negative control, divides four times every three circumferential goat skins down and muscle descend in multiple spots injection cumulative volumes be 5 milliliters of goat-anti small white mouse IgG
1Got blood 700-1000 milliliter in the 66th day from goat arteria carotis, centrifuging and taking serum is used for purifying antibody, its purification process: the sodium chloride concentration in the above-mentioned serum is transferred to 3.3 moles, and add 1 mole of sodium borate of 1/10 volume, above-mentioned solution is joined in albumin A-SepharoseCL4B post, twice each with 3 moles sodium chloride of 10 times of volumes, 50 mMs, pH value are the albumen in 3.0 the glycine solution wash-out post; Add 1 mole of 50 microlitres, pH value in the collection tube and be 8.0 Tris-HCL, collect eluent with it then, after dialysis, be diluted to the 1.2-1.5 mg/ml with the phosphoric acid normal saline buffer solution.
The present invention compared with prior art, its beneficial effect is: be not difficult to find out by above-mentioned solution, without any need for instrument and equipment, it detects the place can store up the place at sample to aflatoxin fast detecting apparatus of the present invention, and only needs just can obtain a result in 5-10 minute when detecting; In addition, it is very simple to operate, as long as is granularity that 20 purpose samples are put into 60% methanol aqueous solution, containing 4% sodium chloride mixes, in the sample cell of test paper lower end, splash into the mixed liquor of 90-100 microlitre then near the plain end that fills up of the polyester fiber of the attached collaurum-monoclonal antibody of spray, then in 5-10 minute, will show the color of reacting aflatoxin content at spray cloth aflatoxin-bovine serum albumin(BSA) complex place, this operation all can be carried out by professional's operation or general personnel, also having a beneficial effect is exactly that it detects required expense and is significantly less than prior art, because can produce in enormous quantities and can long term storage as the main matter-monoclonal antibody of pick-up unit of the present invention.Be fit to the requirement of large-scale industrial production.The accuracy of detection of pick-up unit of the present invention is close with liquid phase chromatography of the prior art.With aflatoxin pick-up unit of the present invention the corn that contains aflatoxin that is provided by U.S. North Carolina Na Zhou state university is detected, its result is extremely close, as shown in the table to the measured result of identical sample with liquid phase chromatography with this university
| Sequence | Extension rate | Testing result of the present invention (estimation) | Liquid phase chromatography |
| ?31A | ?2000 | ?10000ppb | ?11060ppb |
| ?31B | ?2000 | ?12000ppb | ?13740ppb |
| ?31C | ?2000 | ?12000ppb | ?12670ppb |
| ?32A | ?1000 | ?8000ppb | ?6867ppb |
| ?32B | ?2000 | ?12000ppb | ?12558ppb |
| ?32C | ?2000 | ?10000ppb | ?10620ppb |
| ?32D | ?1000 | ?6000ppb | ?6320ppb |
| ?32E | ?1000 | ?5000ppb | ?6262ppb |
| ?32F | ?2000 | ?40000ppb | ?40817ppb |
| ?33A | ?2000 | ?40000ppb | ?40819ppb |
| ?33B | ?2000 | ?40000ppb | ?40156ppb |
| ?33C | ?1000 | ?6000ppb | ?6121ppb |
| ?34A | ?500 | ?1000ppb | ?1260ppb |
| ?34B | ?1000 | ?4000ppb | ?3910ppb |
| ?35A | ?1000 | ?5000ppb | ?6130ppb |
| ?35C | ?200 | ?800ppb | ?680ppb |
| ?35D | ?500 | ?2500ppb | ?3010ppb |
| ?35E | ?500 | ?2500ppb | ?2840ppb |
| ?35F | ?500 | ?2500ppb | ?2800ppb |
In addition, the aflatoxin sample detection of being carried out with liquid phase chromatography by Dalian Product Quality Monitoring Detection Office is compared the very approaching ideal of result with the result that aflatoxin fast detecting apparatus of the present invention detects; Its examining report shows that aflatoxin fast detecting apparatus of the present invention detects aspergillus flavus poison AFB in the corn flour
1Content be 10 nanometer/milliliters, Dalian Product Quality Monitoring Detection Office is 10.6138 nanograms/milliliter with the assay of the same sample of liquid phase chromatography.
Description of drawings
The accompanying drawing drawing is described as follows:
Fig. 1 is the front elevation of aflatoxin pick-up unit;
Fig. 2 is the vertical view of Fig. 1, (1-1), (1-2) are filter paper among the figure, (2) be the polyester fiber pad, (21) for being sprayed on the collaurum-monoclonal anti nanocrystal composition on the polyester fiber pad (2), (3) be nitrocellulose filter, (31) for spraying the aflatoxin-bovine serum albumin(BSA) complex that is attached on the nitrocellulose filter (3), (32) are for spraying the goat-anti small white mouse IgG that is attached on the nitrocellulose filter (3)
1Antibody, (5) base plate for making with plastics.
Embodiment
Now in conjunction with the accompanying drawings (embodiment) the invention will be further described: attach wide about 20 millimeters absorbent filters (1-1) at the right-hand member that scribbles the base plate of adhesive sticker (5), to transfer in absorbance under 540 nano wave lengths with liquid spraying machine is that the collaurum-monoclonal antibody spray of the concentration of 0.5-0.8 is attached on the wide 7 millimeters polyester fiber pad (2), and 37 ℃ of oven dry 1 hour, polyester fiber pad (2) is attached on the base plate, and with overlapping 1～2 millimeter of the filter paper (1-1) of right-hand member; With alternate be that the liquid spraying machine of two nozzles of 10 millimeters is the aflatoxin-bovine serum albumin(BSA) complex (31) and the goat-anti small white mouse IgG of 1-2 mg/ml with concentration
1Antibody (32) with the 0.7-1.5 microlitre/centimetre spray cloth amount spray to be attached to width be on 25 millimeters the nitrocellulose filter (3), oven dry is 1 hour under 37 ℃ of conditions, nitrocellulose filter (3) is attached on the base plate, and with the overlapping 1-2 millimeter of polyester fiber pad (2) of right-hand member; The filter paper (1-2) of 20 mm wides is attached on the base plate, and with the overlapping 1-2 millimeter of nitrocellulose filter (3) of right-hand member.The effect of this filter paper (1-2) is to be relatively easy to by polyester fiber pad (2) and nitrocellulose membrane (3) with the fluid sample that tractive splashes on the filter paper (1-1) by overlapping connecting relation.At 20-25 ℃, relative humidity is under the condition below 40%, and the base plate after will attaching with cutting machine is cut into the rectangular of 4-5 millimeter, in the plastic clip of packing into, in the polybag that contains dry bag of then it being packed into and sealing, keeps in Dark Place in 2-25 ℃.
Being prepared as follows of described collaurum-monoclonal anti liquid solution: 1, with aflatoxin-bovine serum albumin(BSA) complex (AFB
1-BSA) being dissolved in phosphoric acid-normal saline buffer solution (PBS), the Fu Shi Freund's complete adjuvant with equal volume mixes then.Inject subcutaneous 50 micrograms of female small white mouse; In 2 whens week, inject 50 micrograms again, but this antigen mixes with incomplete freund adjuvant, and blood sampling carries out antigen titration degree mensuration after three days; The small white mouse of selecting the efficient valency of performance in the time of all around is supplementary immunization once more, this time antigen is dissolved in PBS, take lumbar injection, blood sampling analysis after three days, the small white mouse of tiring high is put to death by disconnected neck, getting dirty cell and NS-1 myeloma cell merges, employing contains 1.25% methylcellulose, 25% calf serum and HAT nutrient culture media (Hypoxanthine-Aminopterin-Thymidine) are used for optionally promoting hybridoma growth at tissue culture medium (TCM)) semisolid culturemedium of selective reagent carries out hybrid cell and cultivates, hybrid cell is mixed in the semifixed nutrient culture media, in 37 ℃ of CO2gas incubator, cultivates; Taking out the hybridoma clone after 7-10 days is transferred in the 96 hole culture plates, (content is that the hypoxanthine H of 136.1 mg/ml and the thymidine T of 38.8 mg/ml are the HT nutrient culture media of 100X containing 10% calf serum and HT nutrient culture media, during use, dilute 100 times of HT nutrient culture media that concentration is 1X) and the RPMI1640 nutrient culture media of antibiotic in cultivate; After three days, take out nutrient solution 10 microlitres, in using AFB
1Carry out enzyme linked immunoassay (ELISA) in 96 orifice plates of-BSA bag quilt; Determine to produce the hybridoma of aspergillus flavus resisting toxin antibody, it was cultivated 3-5 days in the RPMI1640 nutrient culture media, when treating that antibody concentration reaches the 5-10 mg/ml sodium chloride in the above-mentioned nutrient solution (NaCl) concentration is transferred to 3.3 moles, the 1.0 moles of sodium boratees (pH8.9) that add 1/10 volume are checked pH; Above-mentioned solution is added in albumin A-SepharoseCL4B post, when it flows through albumin A-SepharoseCL4B post in conjunction with IgG
1(immunoglobulin G
1) ability be 5 mg/ml wet gels, contain the monoclonal antibody of 20-50 mg/ml in the supernatant of nutrient solution; With 3.0 moles sodium chloride of 10 times of volumes, 50 mM sodium boratees (pH8.9) flushing chromatographic column secondary is with the albumen in glycine solution (pH3.0) the eluted protein A-SepharoseCL4B post of 100 mMs; In collection tube, add 1 mole of Tris-HCL (pH8.0) of 50 microlitres earlier, collect eluent with it then, mix the pH value that makes every arm gently and be neutrality, avoid bubble and foam to take place to prevent albuminous degeneration; Utilize spectrophotometer to measure the protein adsorption method under 280 nano wave lengths, perhaps the Coomassie brilliant blue method confirms that the purified monoclonal antibody of the concentration of immunoglobulin (Ig) is used for colloid in the collection tube---MONOCLONAL ANTIBODIES SPECIFIC FOR; 2,0.1 gram sodium chloraurate is dissolved in 1000 ml deionized water, be heated to 60 ℃, citric acid solution (0.2% citric acid that when stirring, adds 50 milliliters fast, 0.2% tannic acid), be heated to then 95 ℃ 5 minutes, put then to 4 ℃ of preservations, its formed collaurum diameter is the 18-20 nanometer; 3, with the monoclonal anti liquid solution 4 ℃ to distill water dialysis after, take out supernatant after centrifugal 30 minutes through 0.2 micron membrane filtration with 36000 rev/mins, get its a part of solution and do continuous 10 times of dilutions, volume is 1 milliliter, solution with dilution is added to rapid mixing in 5 milliliters of colloidal gold solutions (its pH value transfers to the PI of a little higher than protein liquid) respectively then, left standstill 5 minutes after 1 milliliter of rapid mixing of sodium chloride solution of adding 10% concentration after 1 minute, measure absorbance value respectively, select the solution of minimum absorption value to be used for bag by collaurum; The concentration dilution of selected protein solution joined to make its ultimate density in the collaurum be the 5-10 mcg/ml after 0.5 mg/ml, after the rapid mixing, allow protein adsorption 5 minutes, it is 1% polyvinyl alcohol (PVA) that per 100 milliliters of collaurums add 1 ml concn, to stop nonspecific agglutination takes place; With 10000 rev/mins centrifugal 1 hour, make the bag quilt collaurum be deposited in the pipe low; Supernatant is abandoned in suction, collaurum is suspended among the PBS that contains 1% PEG; Again with 10000 rev/mins centrifugal 1 hour, the not protein of absorption is removed in washing; Suction is abandoned the collaurum that will wrap quilt after the supernatant and is diluted to that absorbance is the concentration of 0.5-0.8 under 540 nano wave lengths, with 0.2 micron membrane filtration degerming, makes collaurum-monoclonal anti liquid solution.
Described aflatoxin-bovine serum albumin(BSA) complex (AFB
1-BSA) the preparation of solution: get and purchase (St.louis.Mo in Sigma company.The U.S.) (its products catalogue number is A-6655 to aflatoxin-bovine serum albumin(BSA) complex, and every mole of BSA is in conjunction with 11 AFB
1Molecule) (PBS, its compound method is 0.01 mole Na with the phosphoric acid normal saline buffer solution
2HPO
40.15 the NaCl of mole PH7.4) is diluted to 1.5 mg/ml.
The preparation of described goat-anti small white mouse IgG1 antibody-solutions; To be diluted to 10 mg/ml from the small white mouse immunoglobulin (Ig) of U.S. Sigma company, add isopyknic Fu Shi Freund's complete adjuvant again, mix, got blood sample before the immunity of 3-5 milliliter on one's body from carry out goat that adaptability raises through animal feeding room, stay its serum as negative control, again to the small white mouse IgG of 5 milliliters of the muscle of goat or subcutaneous injections
1, appended the small white mouse IgG of above-mentioned same dosage on the 21st day again
1, injected the small white mouse IgG of above-mentioned same dosage on the 42nd day for the third time
1Blood sampling 3-5 milliliter carries out titration after the 45th day, carried out the small white mouse IgG1 of the 4th same dosage on the 63rd day, after the 66th day blood sampling 3-5 milliliter carries out titration, after 10 days from get blood 700-1000 milliliter by the arteria carotis of sheep, centrifuging and taking serum is used for purifying antibody, its purification process: the sodium chloride concentration in the above-mentioned serum is transferred to 3.3 moles, and add 1 mole of sodium borate of 1/10 volume, above-mentioned solution is joined in albumin A-SepharoseCL4B post, twice each sodium chloride of 3 moles with 10 times of volumes, the albumen in the glycine solution of 50 mMs (PH3.0) the wash-out post; Add 1 mole of Tris-HCL (PH8.0) of 50 microlitres in the collection tube, collect eluent with it then, after dialysis, (the PBS its preparation method is 0.01 mole of Na with the phosphoric acid normal saline buffer solution with purified antibody
2HPO
40.15 mole NaCl PH7.4) is diluted to the 1.2-1.5 mg/ml.
The using method of apparatus of the present invention is as follows: detected sample is pulverized be 20 orders and get 20 gram powder and put into the extract of 25-60 milliliter (per 100 milliliters contain 60 ml methanol, 40 ml distilled waters, 4 gram sodium chloride) through fully mixing 5 minutes, leave standstill gentle aspiration supernatant after 10-20 minute again, mix with sample buffer in 1: 3 ratio, apparatus of the present invention placement is 30 degree tilts, make the polyester fiber pad that posts collaurum-monoclonal antibody be in the lower, slowly add the above-mentioned mixed liquor of 90-100 microlitre on the filter paper below the polyester fiber pad, after leaving standstill 5 minutes, observe the color at complex 31 places be sprayed with aflatoxin-bovine serum albumin(BSA) and compare with standard aflatoxin sample (purchasing Sigma company) in the U.S..Can estimate the concentration that contains aflatoxin in the sample.
Claims (2)
1, aflatoxin fast detecting apparatus, it is characterized in that: from one end to the other side overlapping mutually successively on the rectangular base plate of plastics (5) attaches filter paper (1-1), the plain pad of polyester fiber (2), nitrocellulose filter (3) and filter paper (1-2), the attached one deck of spray absorbance under 540 nano wave lengths is that antibody complex (21) falls in the collaurum-Dan Ke of the concentration of 0.5-0.8 on the plain pad of described polyester fiber, monoclonal antibody is wherein produced by hybridoma and selectivity identification aflatoxin, and this hybridoma is via aflatoxin AFB
1-BSA albumen composition immunity small white mouse, obtain by the method for Davis again, purified monoclonal antibody is coated on and makes collaurum-monoclonal anti nanocrystal composition on the collaurum of being made by gold chloride, its particle size is the 18-20 nanometer, be peony, absorbance is the concentration of 0.5-0.8 under 540 nano wave lengths; On described nitrocellulose filter, laterally spray attached following two kinds of materials separately: concentration be the spray plane amount of 1～2 mg/ml be 0.7～1.5 microlitre/centimetre aflatoxin-bovine serum albumin(BSA) complex (31), wherein molecular weight is that 66200 daltonian each bovine serum albumin molecule combine with 11-14 aflatoxin molecule; Concentration is the 1-2 mg/ml, spray cloth amount be 0.7～1.5 microlitre/centimetre goat-anti small white mouse IgG
1Antibody (32), it is by injection small white mouse immunoglobulin (Ig) in the goat body, get its serum that contains antibody and purified after make, its molecular weight is 80.000 dalton, on polyacrylamide gel electrophoresis, present two bands, article one, be that molecular weight is 55000 dalton, another is that molecular weight is 25.000 dalton.
2, the manufacture method of aflatoxin fast detecting apparatus is characterized in that:
A, attach filter paper (1-1) at the right-hand member that is scribbling the base plate of adhesive sticker (5), with transferring in absorbance under 540 nano wave lengths is that collaurum-monoclonal anti nanocrystal composition (21) spray of the concentration of 0.5-0.8 is attached on the polyester fiber pad (2), the polyester fiber pad is attached on the base plate (5), and with the overlapping 1-2 millimeter of filter paper (1-1) of right-hand member; With concentration is the aflatoxin-bovine serum albumin(BSA) complex (31) and the goat-anti small white mouse IgG of 1-2 mg/ml
1Antibody (32) with the 0.7-1.5 microlitre/centimetre spray cloth amount, the spray of the alternate 8-10 of being divided into mm distance is attached on the nitrocellulose filter (3), it is attached on the base plate (5), and with the overlapping 1-2 millimeter of polyester fiber pad (2) of right-hand member; Filter paper (1-2) is attached on the base plate (5), and with the overlapping 1-2 millimeter of nitrocellulose filter (3) of right-hand member; The base plate (5) that posts above-mentioned substance is cut into the rectangular of 4-5 millimeter;
The preparation of B, described collaurum-monoclonal antibody (21) solution: the method for pressing Davis is with aflatoxin-bovine serum albumin(BSA) complex AFB
1-BSA is dissolved among the phosphoric acid physiological saline PBS and Fu Shi Freund's complete adjuvant with volume, get 150 micrograms, divide and be injected in the female small white mouse body for three times, per injection 50 micrograms, in two weeks of space, 3～5 days after the injection are got the disconnected neck of the high small white mouse of tiring and are put to death and get spleen cell and NS-1 myeloma cell's fusion for the third time; Put it into and contain 1.25% methylcellulose, in 37 ℃ incubator, cultivate in 25% the calf serum and the semisolid culturemedium of nutrient culture media, take out after 7-10 days and put into the RPMI1640 nutrient culture media that contains 10% calf serum HT nutrient culture media and antibiotic again and cultivate; Take out after three days, and get 10 microlitres, in using AFB
1Carry out enzyme linked immunoassay ELISA in 96 orifice plates of-BSA bag quilt, determine to produce the hybridoma of aspergillus flavus resisting toxin antibody, the hybridoma of the generation aspergillus flavus resisting toxin obtained was cultivated 3-5 days in the PRMI1640 nutrient culture media, when treating that antibody concentration reaches the 5-10 mg/ml, sodium chloride concentration in the above-mentioned nutrient solution is transferred to 3.3 moles, and add 1 mole of sodium borate of 1/10 volume, above-mentioned solution is joined in albumin A-SepharoseCL4B post, twice each sodium chloride of 3 moles with 10 times of volumes, the sodium borate flushing chromatographic column of 50 mMs is used 100 mMs, pH value is the albumen in 3.0 the glycine solution wash-out post; Add 1 mole of 50 microlitres, pH value in the collection tube and be 8.0 Tris-HCL, collect eluent with it then, the monoclonal antibody of collection is used for collaurum-MONOCLONAL ANTIBODIES SPECIFIC FOR after dialysis; Get 0.1 gram sodium chloraurate and be dissolved in 1000 milliliters the deionized water, heating is boiled to 60 ℃, put into 50 milliliters of 1% sodium citrate solutions of newly joining, wherein contain citric acid 0.2%, contain tannic acid 0.2%, be heated to then 95 ℃ 5 minutes, formed colloid gold particle diameter is the 18-20 nanometer, monoclonal antibody after the dialysis is centrifugal 30 minutes through 36000 rev/mins, get its supernatant through 0.2 micron membrane filtration, join in the colloidal gold solution after its concentration transferred to 0.5 mg/ml, make its ultimate density reach the 5-10 mcg/ml, the polyvinyl alcohol (PVA) PEG that adds 1 milliliter of 1% concentration after 5 minutes in per 100 milliliters of above-mentioned solution to be stoping nonspecific agglutination, with 10000 rev/mins centrifugal 1 hour, remove supernatant, centrifugal 1 hour with 10000 rev/mins again, remove the not protein of absorption, the collaurum of bag quilt is diluted to absorbance is the concentration of 0.5-0.8 under 540 nano wave lengths, go bacterium with 0.2 micron membrane filtration;
The protein complex AFB of C, described aflatoxin-cow's serum
1The preparation of-BSA solution: dilute aflatoxin-bovine serum albumin(BSA) complex to the 1.2-1.5 mg/ml with phosphoric acid normal saline buffer solution PBS;
D, described goat-anti small white mouse IgG
1The preparation of solution: the small white mouse immunoglobulin (Ig) is diluted to 10 mg/ml, adds isopyknic Fu Shi Freund's complete adjuvant again; From carry out through animal feeding room goat that adaptability raises get 3～5 milliliters of immunity on one's body before blood sample make negative control, divides four times every three circumferential goat skins down and muscle descend in multiple spots injection cumulative volumes be 5 milliliters of small white mouse IgG
1Got blood 700-1000 milliliter in the 66th day from goat arteria carotis, centrifuging and taking serum is used for purifying antibody, sodium chloride concentration in the above-mentioned serum is transferred to 3.3 moles, and add 1 mole of sodium borate of 1/10 volume, above-mentioned solution is joined in albumin A-SepharoseCL4B post, twice each sodium chloride of 3 moles with 10 times of volumes, 50 mMs, pH value is the albumen in 3.0 the glycine solution wash-out post, add 1 mole of 50 microlitres in the collection tube, pH value is 8.0 Tris-HCL, collect eluent with it then, after dialysis, be diluted to the 1.2-1.5 mg/ml with the phosphoric acid normal saline buffer solution.
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| GB0702489D0 (en) * | 2007-02-09 | 2007-03-21 | Univ Greenwich | Solid phase extraction of aflatoxins |
| CN101059517B (en) * | 2007-06-06 | 2011-05-11 | 长沙市产商品质量监督检验所 | Method for checking aflatoxin B1 in agricultural by-product |
| CN101930006B (en) * | 2010-08-05 | 2012-08-22 | 中国农业科学院油料作物研究所 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
| CN101900728A (en) * | 2010-08-05 | 2010-12-01 | 中国农业科学院油料作物研究所 | Multi-test-line immunochromatographic test strip for semi-quantitatively detecting aflatoxin B1 and preparation method thereof |
| 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 |
| CN102955031B (en) * | 2011-08-31 | 2015-07-01 | 北京勤邦生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same |
| CN103160472B (en) * | 2011-12-09 | 2014-09-17 | 北京中检维康技术有限公司 | Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit |
| CN103157439B (en) * | 2011-12-09 | 2015-01-21 | 山东出入境检验检疫局检验检疫技术中心 | Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof |
| CN103191589B (en) * | 2013-02-20 | 2014-12-10 | 东南大学 | Preparation method for nano-fluid based on specific absorbance |
| CN110940811A (en) * | 2019-12-05 | 2020-03-31 | 江苏维赛科技生物发展有限公司 | Detection card for simultaneously and quantitatively detecting two toxins in flour as well as preparation and application thereof |
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