CN203535047U - Colloidal gold reagent plate for detecting zearalenone in feed - Google Patents
Colloidal gold reagent plate for detecting zearalenone in feed Download PDFInfo
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- CN203535047U CN203535047U CN201320335935.6U CN201320335935U CN203535047U CN 203535047 U CN203535047 U CN 203535047U CN 201320335935 U CN201320335935 U CN 201320335935U CN 203535047 U CN203535047 U CN 203535047U
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Abstract
The utility model relates to a colloidal gold reagent plate for detecting zearalenone in a feed. The colloidal gold reagent plate consists of a bottom plate, a backing and a cover plate which are superposed from bottom to top in sequence, wherein a sample pad, a colloid gold combination pad, a nitrocellulose membrane and a water absorption pad are tightly adhered to the backing in sequence. The nitrocellulose membrane is provided with a detection line and a control line in sequence in a sprayed manner in the direction from the sample pad to the water absorption pad, so that results are characterized by colors which is visible by the naked eyes. A sample application hole and an observing window are additionally formed in the cover plate, wherein the observing window is divided into a detection area and a control area. The reagent plate disclosed by the utility model can carry out semi-quantitative intuitive detection on the content of the zearalenone in the feed by utilizing an immunity colloid gold process, and is suitable for being used for carrying out large-scale screening on feed samples in feed producing and processing enterprises, inspection and quarantine institutions, and the like.
Description
Technical field
The utility model relates to a kind of gold-immunochromatographyreagent reagent for assay plate, and more specifically, the utility model relates to a kind of colloidal gold reagent plate that detects zearalenone in feed.
Background technology
Zearalenone (zeara lenone, ZEN) claim again F-2 toxin, first from have the corn of head blight, separation obtains, and its toxigenic bacterium is the bacterium dwarf of Fusarium, mainly by Fusarium graminearum, produced, the multiple sickle-like bacteria such as Fusarlum roseum, fusarium tricinctum also can produce this toxin.Zearalenone has multiple derivant, as 7-dehydrogenation zearalenone, Gibberella zeae olefin(e) acid etc.The thermotolerance of zearalenone is stronger, all survivable in food or feed process, mainly pollutes the grain cereal crops such as corn, wheat, barley.
Research shows, zearalenone and metabolin thereof have oestrogen-like hormone effect, cause the hyperfunction disease of animal generation estrogen, causes infertile or miscarriage; Can cause animal acute and chronic poisoning, cause the toxicity symptom of central nervous system, as feel sick, feel cold, headache, mind depression and incoordination etc., even dead.To animal farm, cause tremendous economic loss.In addition, a lot of data show that ZEA has reproductive development, immunotoxicity, hepatotoxicity wind agitation, and potential carcinogenicity etc., current research shows that it to female mammary gland tumour, certain influence occurs also to have.Zearalenone pollutes after feed and food, not only makes agricultural economy be had a strong impact on, but also can bring serious food-safety problem, threatens the mankind's health.
At present, the poisoning specific drug that there is no of animal zearalenone is treated, harm humans and animals being produced in order to control ZEN, must detect zearalenone content in feed, cereal etc., determines whether to contain zearalenone.In European Union's regulation piglet diet, the highest the limiting the quantity of of ZEN is 100 μ g/kg; The content that Italy is defined in ZEN in cereal and cereal product can not surpass 100 μ g/kg; In China's GB13078.2-2006 forage health standard, in regulation feed, limiting the quantity of of zearalenone is 500 μ g/kg, and in GB2761-2011 food security national Specification food, to limit the quantity of be 60 μ g/kg to zearalenone.
At present, more conventional ZEN detection method has thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), vapor-phase chromatography-mass spectroscopy (GC-MS), bioassay method and immunological detection etc.Thin layer chromatography is a kind of method early detecting for toxin, and operating personnel are without process special training, and cost is low, without expensive instrument.But the required reagent of the method is various, and sense cycle is long, sensitivity is not high, far can not meet modern measure requirement.The instrument analytical method such as high performance liquid chromatography, vapor-phase chromatography-mass spectroscopy has that accuracy is high, sensitivity is strong, can microdetermination etc. advantage, and vapor-phase chromatography-mass spectroscopy can detect different fusarium toxins simultaneously, is suitable for the quantitative test of ZEN in feed.But apparatus expensive, to the having relatively high expectations of toxin purity in sample, cause that testing cost is high, the cycle is long, cannot meet the needs of batch samples rapid screening, so use, be restricted.Although it is very pure that microbiological method sample to be checked does not need, method selectivity is poor, and sensitivity is low, is mainly used in qualitative, general only as the evidence of chemical analysis.
ELISA method is one of immunological detection method, has feature special, quick, highly sensitive and that cost is low, is applicable to screening and the generaI investigation of a large amount of samples of mechanism of basic unit.But the method need contact ZEN standard items, be unfavorable for protecting operator's health, and the easy temperature influence of the activity of enzyme, so reagent needs low-temperature preservation, life-span short, and easily cause measurement result repeatability poor.
Utility model content
The above-mentioned defect existing for overcoming prior art, the utility model, for detection demand, has been developed a kind of colloidal gold reagent plate and detection kit that detects zearalenone, by colloidal gold immunization, detects the zearalenone in feed.Agent plate detectability meets the requirements, favorable reproducibility, detection time is short, is applicable to field quick detection, and in testing process without expensive instrument and equipment, also, without using zearalenone standard items, be conducive to protect operator.
To achieve these goals, the technical solution adopted in the utility model is: a kind of colloidal gold reagent plate that detects zearalenone in feed, comprise base plate, backing and cover plate, sample pad, collaurum pad, nitrocellulose filter and the adsorptive pads on described backing, closely pasted successively, between adjacent each several part, have that 1-2mm's is overlapping, wherein, the antibody of the coated colloid gold label of described collaurum pad; The coated specific binding antigen of described nitrocellulose filter and polyclonal antibody.
Preferably, described base plate, backing and cover plate are from top to bottom compressed to one successively.
Preferably, described nitrocellulose filter is sprayed with detection line and control line from sample pad to adsorptive pads direction successively, and described detection line is coated with zearalenone-BSA specific binding antigen, and described control line is coated with sheep anti mouse polyclonal antibody.
Preferably, the particle size of described colloid gold particle is about 30nm, and the antibody of described colloid gold label is zearalenone monoclonal antibody.
Preferably, described sample pad is made by glass fibre, and described collaurum pad is made by polyester film, and described adsorptive pads is filter paper.
Preferably, described base plate and cover plate are two plastic formworks, and well and view window are set on described cover plate, and the inhomogeneous distribution of described well and view window on the cover board, between described well and view window, have interval, described view window is divided into detection zone and control zone.
The utility model agent plate application chromatography type antibody mediated immunity competition principle, by antigen and golden labeling antibody reaction solution, zearalenone content in specific detection Feed Sample, if contain zearalenone in sample solution, zearalenone is the zearalenone monoclonal antibody combination first and on colloid gold particle just, and cannot be combined by the zearalenone specific antigen on detection line.When the zearalenone content in sample equals or exceeds agent plate detection limit, the detection line colour developing in agent plate is shallow even without colour developing compared with control line, is judged to be the positive.Otherwise zearalenone content is below agent plate detection limit or during noresidue in sample, the detection line colour developing in agent plate is close with control line or partially dark, is judged to be feminine gender.
The utility model agent plate is comprised of base plate, backing and cover plate, is closely pasting successively sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on backing.Base plate, backing and cover plate are from top to bottom compressed to one successively.
On collaurum pad, be coated with the zearalenone monoclonal antibody of colloid gold label; On nitrocellulose filter, from sample pad to adsorptive pads direction, be coated with successively zearalenone-BSA specific binding antigen and sheep anti mouse polyclonal antibody IgG, respectively as detection line and control line.The carrier protein of coupling zearalenone can be bovine serum albumin(BSA), ovalbumin, hemocyanin etc.Well and view window are set on cover plate, and the inhomogeneous distribution of well and view window on the cover board, has interval between well and view window, and view window is divided into detection zone and control zone.
Each several part constituent and the function of the utility model agent plate are as follows:
Base plate, cover plate, be two plastic formworks, plays fixedly backing and sign each functional areas (well, detection zone, control zone).
Backing, is made by the toughness material that do not absorb water that simultaneously scribbles adhesive sticker, plays other ingredients of fixed support agent plate.
Sample pad, is made by glass fibre, works to absorb sample solution and buffering sample solution pH value.
Collaurum pad, is made by polyester film, has the bond of anti-zearalenone monoclonal antibody and colloid gold particle on it, for the reaction of effective constituent in sample solution and golden labeling antibody provides place.
Nitrocellulose filter, is sprayed with detection line and control line from sample pad to adsorptive pads direction, by reaction result with macroscopic characterization out successively.
Adsorptive pads, is made by filter paper, and solution unnecessary in course of reaction is absorbed.
The utility model agent plate has following beneficial effect:
(1) specificity is good.The utility model agent plate is 100% to the cross reacting rate of zearalenone, to the cross reacting rate of the mycotoxins such as vomitoxin, fumonisins, ochratoxin A all lower than 1%.Visible, to zearalenone, reaction has height selectivity to the utility model agent plate.
(2) highly sensitive.The utility model agent plate is 100 μ g/kg to the minimum detectability of zearalenone in feed, meets country's requirement of limiting the quantity of, and in to feed, in the fast detecting field of zearalenone, has reached more excellent level.According to reality, detect demand, by the sample pretreatment process detectability of the different capable of regulating agent plate of solution, special-purpose dilution consumption at the middle and upper levels, specifically in Table 1, be applicable to all kinds of enterprises and testing agency.
Table 1 zearalenone immune colloid gold quick detection reagent plate detection limit reference table
(3) simple to operate, quick, safety.The utility model agent plate is incorporated into the required most of raw material of reaction in PVC backing, drip after sample, antigen-antibody reaction is carried out fast on immobilon-p, greatly shorten the sample time, and sample, after a sample 3-5 minute can be with the naked eye by the detection line on judgement nitrocellulose filter and the shade reading result of control line without special processing.Detect implementation process and do not rely on any experimental facilities, without toxin standard model, ordinary person all can operate.
(4) cost is low, easily promotes.The utility model agent plate production technology is simple, and flow process is ripe.Low production cost, requires less investment while yielding quicker results.
Accompanying drawing explanation
Fig. 1 is zearalenone immune colloid gold quick detection reagent backboard lining structure schematic diagram, and wherein 1 is sample pad, and 2 is collaurum pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is control line, and 6 is adsorptive pads, and 7 is adhesive sticker, and 8 is PVC base plate.
Fig. 2 is zearalenone immune colloid gold quick detection reagent plate operation chart, and wherein S is well, and C is control zone, and T is detection zone.
Fig. 3 is that the zearalenone immune colloid gold quick detection reagent fruit of hardening is judged negative schematic diagram; Fig. 4 is that the zearalenone immune colloid gold quick detection reagent fruit of hardening is judged positive schematic diagram; Fig. 5 be zearalenone immune colloid gold quick detection reagent harden fruit judge invalid schematic diagram, wherein C is control zone, T is detection zone.
Embodiment
The preparation of the utility model agent plate comprises the preparation of carrier protein couplet thing, the preparation of anti-zearalenone monoclonal antibody, the preparation of colloidal gold solution, the assembling of the preparation of the anti-zearalenone monoclonal antibody of colloid gold label and zearalenone immune colloid gold quick detection reagent plate.
1. the coupling of haptens and carrier protein
The synthetic method of zearalenone-6-carboxylic methoxy oxime: the ZEN of 5mg is dissolved in 4mL pyridine, adds the O-ethyloic azanol of people 3mg, at room temperature stirs 24h, vacuum drying afterwards, and water-soluble solution, adjusts pH to 8, and ethyl acetate extracts 3 times, anhydrates, crystallization.
Utilize carbodlimide method to synthesize zearalenone-bovine serum albumin conjugate (ZEN-BSA): crystal (zearalenone-6-carboxylic methoxy oxime) is dissolved in and in dioxane, is made into 5mmol/L solution, take 30mg BSA and be dissolved in 2mL, in the PBS of 0.05mol/L (pH7.4).At 4 ℃, the two is mixed, more slowly drip the solution that the N-hydroxy succinic acid imines (NHS) of 3mg and the N of 6mg, N '-bis-hexamethylene carbodiimide (DCC) are dissolved in 0.5mL dioxane, potpourri is stirring reaction 24h under room temperature, dialysis, centrifugal, freezing standby.
With ovalbumin (OVA), substitute BSA, adopt the synthetic zearalenone-ovalbumin conjugate (ZEN-OVA) of same method.
2. the preparation of anti-zearalenone monoclonal antibody
Animal immune: select female BALB/c mouse in 6-8 age in week, get 50 μ g comlete antigens, with Freund's adjuvant emulsification, only carry out just exempting from neck and subcutaneous multi-point injection by 200 μ L/.Immunity afterwards changes incomplete Freund's adjuvant into, and every immunity in 2 weeks once, dosage is identical, lumbar injection.4 exempt from rear 10d blood sampling measures, and selects serum titer height and ZEN to suppress strong mouse.3d comlete antigen abdominal cavity booster immunization before merging.
Fusion of Cells: 1d before merging, under aseptic condition, to get BALB/c mouse abdominal cavity inner cell and make feeder cells, paving 96 porocyte culture plates are standby; The de-lethal mouse of neck, the aseptic spleen of getting is prepared splenocyte, according to immune spleen cell and 10: 1 cell mixings of SP2/0 myeloma cell, with 50% polyglycol (PEG)-1500 is short, melts; Cell suspension inoculation after merging, to being covered with in 96 porocyte culture plates of feeder cells, is successively cultivated with HAT and HT nutrient solution, changed liquid respectively at the 4th day and the 7th day, within the 12nd day, reinstate the cultivation of DMEM complete culture solution.
In body, induce ascites: the whiteruss 0.5mL/ of the BALB/c mouse lumbar injection sterilizing of approximately 8 weeks only, after 10d, with method injection monoclonal positive cell 0.2mL/ only, after 8-10d, extract ascites, centrifugal removing after precipitation of grease, obtain mouse ascites, with saturated ammonium sulfate salting out method, purify ascites.
3. the preparation of colloidal gold solution
The mean size of colloid gold particle is 30nm, and its preparation method, for to add 1mL1% trisodium citrate in 100mL deionized water, boils the rear 1mL1% gold chloride that adds rapidly, continues to boil 10min, cooling after, save backup at 4 ℃.
4. the preparation of colloid gold label zearalenone monoclonal antibody
Get the 100mL colloidal gold solution having prepared, with 0.1mol/L solution of potassium carbonate, adjust pH to 8.0.Add while stirring the anti-zearalenone monoclonal antibody of 1.5mg, stir 20min, more dropwise add 2mL25mol/L PEG 20000 (PEG20000), stir 15min.The centrifugal 15min of 20,000rpm, abandons supernatant, adds 10mL pH7.4PBS damping fluid (containing 0.4mol/L PEG) to clean 2 times.Precipitation is dissolved containing the PBS damping fluid (pH7.4) of 2%BSA with 5mL, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ save backup.
5. the assembling of zearalenone immune colloid gold quick detection reagent plate
With reference to Fig. 1, with some film machine, the carrier protein couplet thing of debita spissitudo and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and control line, 37 ℃ of oven drying 8h.In kind, the colloid gold label zearalenone monoclonal antibody preparing is coated on collaurum pad.
Detecting reagent set becomes PVC backing, is stained with in order sample pad, collaurum pad, nitrocellulose filter and adsorptive pads thereon.With cutting machine, the kilocalorie posting is cut into the wide bar of 4mm, pack into and in plastic formwork, make detection agent plate, then put into the aluminium foil bag sealed storage with drying agent.
6. zearalenone immune colloid gold quick detection reagent plate detects implementation and operation method
6.1 sample preparation
Take 1g and grind sample in 5mL centrifuge tube; Add 4mL extraction agent, concuss is after 3 minutes, and centrifugal 5 minutes of 4,000rpm is divided into two-layerly, and upper strata is the solution of clarification; According to required detection limit, get corresponding upper solution and special-purpose dilution consumption; After piping and druming mixes, to be checked.
6.2 using method
From packaging bag, take out agent plate, draw sample solution 100 μ L to be checked and be added drop-wise in well, after application of sample, start timing; Result should read at 3-5min, and other times interpretation is invalid.During observation, agent plate is placed horizontally at observer front.
6.3 result judgements
In view window, T line colour developing than C line deeply or equally dark negative, represents in sample that zearalenone content is lower than detection limit or containing zearalenone.As shown in Figure 3.
In view window, T line colour developing is more shallow or T line is positive without colour developing than C line, represents that in sample, zearalenone content is equal to or higher than detection limit.As shown in Figure 4.
In view window, not occurring C line, may be that misoperation or agent plate lost efficacy.Should again read instructions, and retest by new agent plate.As shown in Figure 5.
Claims (6)
1. a colloidal gold reagent plate that detects zearalenone in feed, it is characterized in that, comprise base plate, backing and cover plate, sample pad, collaurum pad, nitrocellulose filter and the adsorptive pads on described backing, closely pasted successively, between adjacent each several part, have that 1-2 mm's is overlapping, wherein, the antibody of the coated colloid gold label of described collaurum pad; The coated specific binding antigen of described nitrocellulose filter and polyclonal antibody.
2. detect as claimed in claim 1 the colloidal gold reagent plate of zearalenone in feed, it is characterized in that, described base plate, backing and cover plate are from top to bottom compressed to one successively.
3. detect as claimed in claim 1 the colloidal gold reagent plate of zearalenone in feed, it is characterized in that, described nitrocellulose filter is sprayed with detection line and control line from sample pad to adsorptive pads direction successively, described detection line is coated with zearalenone-BSA specific binding antigen, and described control line is coated with sheep anti mouse polyclonal antibody.
4. detect as claimed in claim 1 the colloidal gold reagent plate of zearalenone in feed, it is characterized in that, the antibody of described colloid gold label is zearalenone monoclonal antibody.
5. detect as claimed in claim 1 the colloidal gold reagent plate of zearalenone in feed, it is characterized in that, described sample pad is made by glass fibre, and described collaurum pad is made by polyester film, and described adsorptive pads is filter paper.
6. detect as claimed in claim 1 the colloidal gold reagent plate of zearalenone in feed, it is characterized in that, described base plate and cover plate are two plastic formworks, well and view window are set on described cover plate, the inhomogeneous distribution of described well and view window on the cover board, between described well and view window, have interval, described view window is divided into detection zone and control zone.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105334319A (en) * | 2015-10-21 | 2016-02-17 | 黑龙江省乳品工业技术开发中心 | Test strip for detecting zearalonone in milk |
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2013
- 2013-06-13 CN CN201320335935.6U patent/CN203535047U/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105334319A (en) * | 2015-10-21 | 2016-02-17 | 黑龙江省乳品工业技术开发中心 | Test strip for detecting zearalonone in milk |
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Address after: 215437 Jiangsu city of Suzhou province Taicang city Shaxi town Xingang Yuewang Road No. 239 Patentee after: ANYOU BIOTECHNOLOGY GROUP Co.,Ltd. Address before: 215437 Jiangsu city of Suzhou province Taicang city Shaxi town Xingang Yuewang Road No. 239 Patentee before: ANYOU BIOTECHNOLOGY GROUP CO.,LTD. |
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Granted publication date: 20140409 |
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