CN107831320A - A kind of food inspection kit for being used to detect ZER - Google Patents
A kind of food inspection kit for being used to detect ZER Download PDFInfo
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- CN107831320A CN107831320A CN201711324556.6A CN201711324556A CN107831320A CN 107831320 A CN107831320 A CN 107831320A CN 201711324556 A CN201711324556 A CN 201711324556A CN 107831320 A CN107831320 A CN 107831320A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
The invention discloses a kind of food inspection kit for being used to detect ZER, including kit, the kit bottom is provided with bottom, the kit is internally provided with the hole that installed reagents bottle matches, and the reagent included in described hole has concentrated cleaning solution, redissolve liquid, antibody working solution, enzyme marker dilution, terminate liquid, nitrite ion, confining liquid and titer, and the hole side to match is provided with the joint strip of related liquid, match somebody with somebody in the bottom and be set with enzyme frame, abacus is set at the top of the enzyme frame, plate rail is set with the orifice plate, Reagent Tube is arranged with the plate rail, it is easy to operate, detection sensitivity is high, cost is low, the measure that one-time continuous carries out ZER residual quantity in multiple samples can be carried out, reduce the time required for detection sample.
Description
Technical field
The present invention relates to technical field of food detection, specially a kind of food inspection reagent for being used to detect ZER
Box.
Background technology
ZER category female hormone, have and maintain secondary sex character and for protein assimilating effect, in addition to general treatment purposes,
Also it is applied to ruminant as growth promoter, the edible vegetalitas production containing Zeranol residuals is found until the end of the nineties
Product can cause human body sexual performance disorderly and influence the normal development of secondary sex characters, under external condition induction, it is also possible to carcinogenic；Its
After secondary ZER discharges animal body is outer, also secondary pollution and environmental pollution, application number can be caused through drinking-water and food：Application number
201220564064.0 the title of utility model：ZER detection kit, the technical solution that the patent provides
It is：The kit includes solid phase carrier, the recessed bottle position for putting reagent, the reagent bottle on recessed bottle position, is set on solid phase carrier
There is capillary strip, capillary strip includes micropore, coated layer, and coated layer is made up of ZER coupled antigen；Reagent bottle includes being equipped with
The reagent of the reagent bottle of ZER monoclonal antibody solution, sheep anti mouse antiantibody equipped with horseradish peroxidase-labeled
Bottle.
At present in the world in vegetable food ZER residual instrument detection method mainly have liquid-mass chromatography method and
Gas-matter Lian Fafa and liquid chromatography-tandem mass spectrometry method for determining, this quasi-instrument detection method, the degree of accuracy is high, and precision is good, can
As the final confirmation of the residual detection of medicine, but its Sample pretreatment is complicated, detection time is long, needs expensive instrument and equipment, and right
Experiment operator requires high.
The content of the invention
In order to overcome the shortcomings of prior art, the present invention provides a kind of food inspection for being used to detect ZER
Kit, its is easy to operate, and detection sensitivity is high, and cost is low, can carry out one-time continuous and carry out ZER in multiple samples
The measure of residual quantity, reduce the time required for detection sample, effectively can solve the problems, such as in background technology.
The technical solution adopted for the present invention to solve the technical problems is：A kind of food inspection for being used to detect ZER
Test agent box, including kit, the kit bottom are provided with bottom, and the kit is internally provided with the matching of installed reagents bottle
The hole of set, and the reagent included in described hole have concentrated cleaning solution, redissolve liquid, antibody working solution, enzyme marker dilution,
Terminate liquid, nitrite ion, confining liquid and titer, and the hole side to match is provided with the joint strip of related liquid, in the bottom
With enzyme frame is set with, abacus is set at the top of the enzyme frame, plate rail is set with the orifice plate, is arranged with the plate rail
Reagent Tube.
As a kind of preferable technical scheme of the present invention, the coating antigen of the food inspection kit uses ZER
With the antigen of ovalbumin coupling, the antibody of the food inspection kit is using ZER and protein molecule antigen
Monoclonal antibody, and antigenic dilution is 0.05M carbonate buffer solutions, antibody working solution is that 0.05M contains bovine serum albumin and protective agent
Phosphate buffer solution.
As a kind of preferable technical scheme of the present invention, the confining liquid contains 0.5% casein, 1% calf serum,
The phosphate buffer of 5 ‰ Sodium azides, and its configuration concentration difference conduct of calf serum concentration 0.5%, 1%, 2%, 4%, 5% A, B,
C, five kinds of selections of D, E.
As a kind of preferable technical scheme of the present invention, the enzyme marker dilution contains 5% calf serum and protection
The buffer solution of agent, its configuration concentration difference calf serum concentration 2%, 4%, 8%, 16%, 30% is as five kinds of selections of A, B, C, D, E.
It is anti-using sheep anti mouse in the detection as a kind of preferable technical scheme of the present invention, the food inspection kit
Body, and it is attached with blank ox urine sample.
As a kind of preferable technical scheme of the present invention, solution A and peroxidating of the nitrite ion for tetramethyl benzidine
The B solution of hydrogen mixes.
As a kind of preferable technical scheme of the present invention, the terminate liquid uses the 2M concentrated sulfuric acids.
Compared with prior art, the beneficial effects of the invention are as follows：
Kit detection is limited to 0.5ng/ml, and between 70.0%~116.0%, the coefficient of variation is less than TIANZHU XINGNAO Capsul
20%, it can be preserved 3 months under the conditions of 2~8 DEG C, the whole system reaction time is：130 minutes, compare instrumental method（Gas phase, liquid
Phase method etc.）Many times are saved, select compound zearelone, α-corn in structure and as zearalenones
Red mould enol, ZER, β-zearalenol are reacted as standard items, detect cross reacting rate, kit pair
Zearalenone, zearelone, α-zearalenol, the cross reacting rate of ZER are higher, can be simultaneously
Detection is synchronized to ZER and zearalenone, while red mould alcohol is detected, passes through the intersection of zeranol
Reactivity is mapped the concentration of red mould alcohol, so as to fast and accurately detect.
Brief description of the drawings
Fig. 1 is the overall structure diagram of the present invention；
Fig. 2 is the Elisa plate structure schematic diagram of the present invention；
In figure：1- kits；2- bottoms；3- concentrated cleaning solutions；4- joint strips；5- antibody working solutions；6- nitrite ions；7- terminate liquids；
8- titers；9- enzyme frames；10- plate rails；11- Reagent Tubes；12- orifice plates；13- redissolves liquid；14- enzyme marker dilutions；15- is sealed
Close liquid.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
The explanation of following embodiment is refer to the attached drawing, can be to the specific embodiment implemented to the example present invention.
The direction and position term that the present invention is previously mentioned, for example, " on ", " in ", " under ", "front", "rear", "left", "right", " interior ", "
Outside ", " side " etc., only it is direction and position with reference to annexed drawings.Therefore, the direction and position term used is to illustrate
And understand the present invention, and it is not used to the limitation present invention.
Embodiment：
As depicted in figs. 1 and 2, the invention provides a kind of food inspection kit for being used to detect ZER, including examination
Agent box 1, the bottom of kit 1 are provided with bottom 2, and the kit 1 is internally provided with the hole that installed reagents bottle matches, and
The reagent included in described hole has concentrated cleaning solution 3, redissolves liquid 13, antibody working solution 5, enzyme marker dilution 14, termination
Liquid 7, nitrite ion 6, confining liquid 15 and titer 8, and the hole side to match is provided with the joint strip 4 of related liquid, the bottom 2
It is interior with enzyme frame 9 is set with, the top of enzyme frame 9 sets abacus 12, and plate rail 10, the disk are set with the orifice plate 12
Reagent Tube 11 is arranged with frame 10.
Wherein, the coating antigen of the food inspection kit is using ZER and the antigen of ovalbumin coupling, institute
The antibody of food inspection kit is stated using the monoclonal antibody of ZER and protein molecule antigen, and antigenic dilution is
0.05M carbonate buffer solutions, antibody working solution 5 are that 0.05M contains bovine serum albumin and protectant phosphate buffer solution；It is described
Confining liquid 15 is distinguished containing 0.5% casein, 1% calf serum, the phosphate buffer of 5 ‰ Sodium azides, and its configuration concentration
Calf serum concentration 0.5%, 1%, 2%, 4%, 5% is as five kinds of selections of A, B, C, D, E；The enzyme marker dilution 14 contains 5%
Calf serum and protectant buffer solution, its configuration concentration difference calf serum concentration 2%, 4%, 8%, 16%, 30% as A, B, C,
D, five kinds of selections of E；The food inspection kit uses sheep anti mouse antiantibody in the detection, and is attached with blank ox urine sample；
The nitrite ion 6 mixes for the solution A of tetramethyl benzidine and the B solution of hydrogen peroxide；The terminate liquid 7 is dense using 2M
Sulfuric acid.
ELISA method of the invention based on indirect competition, passes through pre-coated corn on the capillary strip between adjacent reagent pipe
The antigen of red mould alcohol and ovalbumin coupling, residue ZER and Gibberella zeae pre-coated on capillary strip in sample
The antibody of alcohol and the anti-ZER of antigenic competition of ovalbumin coupling, adds enzyme marker dilution 14, is entered with nitrite ion 6
Row tmb substrate develops the color, the content of sample light absorption value and its contained ZER into negative correlation, compared with standard curve multiplied by with
Its corresponding extension rate can draw the content of residue ZER, the determination 1 of most suitable coating concentration:5000, it is most suitable
Monoclonal antibody and ELIAS secondary antibody extension rate are defined as 5000:2500, sample solution and monoclonal antibody liquor capacity ratio are defined as 50：
50, coating buffer is defined as B, and confining liquid is defined as D, and ELIAS secondary antibody dilution is defined as D, and monoclonal antibody dilution is defined as
D, substrate nitrite ion are defined as intermediate concentration；Selected various solution are carried out with compbined test, maximum light absorption value, IC50,
Blank ox urinates measured value, and 21ng/ml addition measured values meet the requirements.It can carry out further testing determination and draw：Coating
Condition is defined as 37 DEG C of incubation 1h, then 4 DEG C are stayed overnight, and off-period is defined as 60min, and the monoclonal antibody reaction time is defined as
60min, ELIAS secondary antibody reaction time are defined as 60min, and substrate color condition is defined as 10min.
The detection of red mould alcohol is being determined by the synchronous cross reaction with zearalenone simultaneously, in the preparation of antigen
On：By ZEN and hydroxylamine hydrochloride condensation reaction, a new hydroxyl is introduced, then react to obtain with carboxyl-functional with succinic anhydride
The haptens of group.The specific steps of haptens:159mgZEN and hydroxylamine hydrochloride be stirred at room temperature 12 in 5.0ml pyridine solutions~
24h, revolving remove pyridine and unreacted azanol, obtain crude product, add 5.0ml tetrahydrofurans and 200 μ l pyridines, room temperature are stirred
0.5h is mixed, the 50mg succinic anhydrides being dissolved in 1.0ml tetrahydrofuran solutions are added dropwise, reacts at room temperature 12~24h, revolving removes solvent
And pyridine, column chromatography purifying, obtain ZEN haptens.
In the preparation of antibody：After ZEN-BSA is mixed with equivalent freund adjuvant, it is immunized in a manner of subcutaneous multi-point injection
Balb/c mouse；Mouse boosting cell is won after immune 4 times to merge with myeloma cell, and screens positive cell strain；To filtering out
Positive cell strain subclone, expanded after purification；Abdomen is carried out to Balb/c mouse with the positive myeloma cell after amplification
Chamber is injected, and is collected peritoneal fluid after 7d, can be obtained ZEN monoclonal antibodies after purification.
In the preparation of coating antigen：ZEN haptens and OVA are coupled to obtain coating antigen.Concrete operations are as follows:Take ZEN half
Antigen 5mg is dissolved with 1.0mlDMF, takes the μ l of isobutyl chlorocarbonate 5 to add, 10 DEG C of stirring reaction 30min, you can obtain reaction solution
A；OVA30mg is weighed, is allowed to be substantially dissolved in 2.0ml50mmol/L sodium carbonate liquors and obtains B liquid；Reaction solution A is slow dropwise
It is added drop-wise in reaction solution B, 10 DEG C of reaction 4h, then stays overnight for 4 DEG C；With 0.01mol/LPBS dialysis 3d, 3 dialyzates are changed daily.
Packing, is saved backup in -20 DEG C.
Double check mode：Coating antigen is diluted into debita spissitudo with coating buffer to add in ELISA Plate, the μ l of 1 hole 100；37℃
2h is incubated, extraction raffinate is outwelled, is washed 1 time with wash operating solution；The μ l of 1 hole of confining liquid 150 are added, 37 DEG C of incubation 2h, are got rid of wherein molten
Liquid, pat dry, 10mg enzymes are dissolved in into room temperature in 0.10mol/LpH6.8PBS of the 0.2ml containing 1.25% glutaraldehyde places 18h, fully thoroughly
Analysis removes unreacted glutaraldehyde with SephadexG25 posts.Physiological saline is added then to add 1.0ml (containing 5mg) to 1.0ml anti-
Liquid solution and 1.0ml1.00mol/LpH9.6 carbonate buffer solutions, 4 DEG C of refrigerators place 24h after mixing, add
0.1ml0.20mol/L lysines, room temperature 2h is put, fully dialysed with 0.15mol/LpH7.2PBS liquid, centrifugation goes to precipitate, supernatant
As enzyme conjugates, further applied with ammonium sulfate precipitation, be made up of A liquid and B liquid after purification, A formula of liquid is to take 1g peroxides
Change urea, 10.3g citric acids, 35.8gNa2HPO412H2O, 100 μ l Tween-20s are dissolved in deionized water, are adjusted to pH=5.0, and
Final to be settled to 1000ml, to take 700mg tetramethyl benzidines, 10.3g citric acids are dissolved in deionized water, are adjusted to pH B formula of liquid
Value=2.6, and is finally settled to 1000ml, to being coated with sample solution or ZEN that the μ l of 1 hole 20 are added in the ELISA Plate of ZEN antigens
Standard liquid (concentration is 0 μ g/ml, 5.0 μ g/ml, 15.0 μ g/ml, 45.0 μ g/ml, 135.0 μ g/ml, 405.0 μ g/ml), and
Add the μ l of 1 hole 100 enzyme labelled antibody working solution, vibration is mixed after reacting 10min in 25 DEG C of light protected environments.Clapped after washing 5 times
It is dry, substrate A liquid, each 50 μ l in the hole of B liquid 1 are added, the μ l of 1 hole of the concentrated sulfuric acid 50 for being 2.00mol/L with concentration after reaction 5min are terminated, and
With ELIASA detection OD value (OD), standard curve, selection are drawn according to the logarithm of percentage absorbance and standard concentration
Compound zearelone, α-zearalenol, ZER, β-corn in structure and as zearalenones
Red mould enol is reacted as standard items, detects cross reacting rate.
In summary, the main characteristic of the invention lies in that：Kit detection is limited to 0.5ng/ml, and TIANZHU XINGNAO Capsul exists
Between 70.0%~116.0%, the coefficient of variation is less than 20%, can be preserved 3 months under the conditions of 2~8 DEG C, when whole system is reacted
Between be：130 minutes, compare instrumental method（Gas phase, liquid phase process etc.）Many times are saved, are selected in structure and Gibberella zeae alkene
Compound zearelone, α-zearalenol, ZER, β-zearalenol enter as standard items as ketone
Row reaction, detects cross reacting rate, as a result shows, kit is to zearalenone, zearelone, α-Gibberella zeae alkene
Alcohol, the cross reacting rate of ZER are higher, can synchronize inspection to ZER and zearalenone simultaneously
Survey, while red mould alcohol is detected, the concentration of red mould alcohol is mapped by the cross reacting rate of zeranol, so as to
Fast and accurately detect.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference in claim should not be considered as to the involved claim of limitation.
Claims (7)
- A kind of 1. food inspection kit for being used to detect ZER, it is characterised in that：Including kit（1）, the examination Agent box（1）Bottom is provided with bottom（2）, the kit（1）It is internally provided with the hole that installed reagents bottle matches, and the hole The reagent included in hole has concentrated cleaning solution（3）, redissolve liquid（13）, antibody working solution（5）, enzyme marker dilution（14）, eventually Only liquid（7）, nitrite ion（6）, confining liquid（15）And titer（8）, and the hole side to match is provided with the joint strip of related liquid （4）, the bottom（2）It is interior with being set with enzyme frame（9）, the enzyme frame（9）Top sets abacus（12）, the orifice plate （12）On be set with plate rail（10）, the plate rail（10）On be arranged with Reagent Tube（11）.
- A kind of 2. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute The coating antigen of food inspection kit is stated using ZER and the antigen of ovalbumin coupling, the food inspection kit Antibody using the monoclonal antibody of ZER and protein molecule antigen, and antigenic dilution be 0.05M carbonate buffer solutions, anti- Body running liquid（5）Contain bovine serum albumin and protectant phosphate buffer solution for 0.05M.
- A kind of 3. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute State confining liquid（15）Containing 0.5% casein, 1% calf serum, the phosphate buffer of 5 ‰ Sodium azides, and its configuration concentration Calf serum concentration 0.5%, 1%, 2%, 4%, 5% is as five kinds of selections of A, B, C, D, E respectively.
- A kind of 4. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute State enzyme marker dilution（14）Containing 5% calf serum and protectant buffer solution, its configuration concentration difference calf serum is dense Degree 2%, 4%, 8%, 16%, 30% is as five kinds of selections of A, B, C, D, E.
- A kind of 5. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute State food inspection kit and use sheep anti mouse antiantibody in the detection, and be attached with blank ox urine sample.
- A kind of 6. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute State nitrite ion（6）The B solution of solution A and hydrogen peroxide for tetramethyl benzidine mixes.
- A kind of 7. food inspection kit for being used to detect ZER according to claim 1, it is characterised in that：Institute State terminate liquid（7）Using the 2M concentrated sulfuric acids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711324556.6A CN107831320A (en) | 2017-12-13 | 2017-12-13 | A kind of food inspection kit for being used to detect ZER |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711324556.6A CN107831320A (en) | 2017-12-13 | 2017-12-13 | A kind of food inspection kit for being used to detect ZER |
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| Publication Number | Publication Date |
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| CN107831320A true CN107831320A (en) | 2018-03-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201711324556.6A Pending CN107831320A (en) | 2017-12-13 | 2017-12-13 | A kind of food inspection kit for being used to detect ZER |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563993A (en) * | 2004-03-25 | 2005-01-12 | 中国农业大学 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
| CN201852836U (en) * | 2010-10-27 | 2011-06-01 | 北京勤邦生物技术有限公司 | ELISA (enzyme-linked immuno sorbent assay) kit for detecting zeranol |
| CN205210092U (en) * | 2015-11-09 | 2016-05-04 | 重庆创生生物科技集团有限公司 | Virogene separation detect reagent box |
| CN106645697A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | A kit for detecting zeranol in foods |
| CN208506058U (en) * | 2017-12-13 | 2019-02-15 | 百奥森（江苏）食品安全科技有限公司 | It is a kind of for detecting the food inspection kit of zearalanol |
-
2017
- 2017-12-13 CN CN201711324556.6A patent/CN107831320A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563993A (en) * | 2004-03-25 | 2005-01-12 | 中国农业大学 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
| CN201852836U (en) * | 2010-10-27 | 2011-06-01 | 北京勤邦生物技术有限公司 | ELISA (enzyme-linked immuno sorbent assay) kit for detecting zeranol |
| CN205210092U (en) * | 2015-11-09 | 2016-05-04 | 重庆创生生物科技集团有限公司 | Virogene separation detect reagent box |
| CN106645697A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | A kit for detecting zeranol in foods |
| CN208506058U (en) * | 2017-12-13 | 2019-02-15 | 百奥森（江苏）食品安全科技有限公司 | It is a kind of for detecting the food inspection kit of zearalanol |
Non-Patent Citations (1)
| Title |
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| 王东等: "饲料中玉米赤霉醇ELISA检测试剂盒的试剂盒应用", 《湖南饲料》 * |
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Application publication date: 20180323 |