CN208506058U - It is a kind of for detecting the food inspection kit of zearalanol - Google Patents

Abstract

The utility model discloses a kind of for detecting the food inspection kit of zearalanol, 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 in described hole including has concentrated cleaning solution, redissolve liquid, antibody working solution, enzyme marker dilution, terminate liquid, developing solution, confining liquid and titer, and the hole side to match is provided with the joint strip of related liquid, match in the bottom and is set with enzyme frame, abacus is set at the top of the enzyme frame, plate rail is set on the orifice plate, Reagent Tube is arranged on the plate rail, it is easy to operate, detection sensitivity is high, it is at low cost, it can carry out the measurement that one-time continuous carries out zearalanol residual quantity in multiple samples, reduce the time required for detection sample.

Description

It is a kind of for detecting the food inspection kit of zearalanol

Technical field

It is specially a kind of for detecting the food inspection of zearalanol the utility model relates to technical field of food detection Kit.

Background technique

Zearalanol category female hormone, with maintenance secondary sex character and for protein assimilating effect, in addition to general treatment purposes, Also it is applied to ruminant as growth promoter, is produced until discovery at the end of the nineties is edible containing the remaining vegetalitas of Zeranol Product can cause the disorder of human body sexual performance and influence the normal development of secondary sex characters, under external condition induction, it is also possible to carcinogenic；Its After secondary ZER discharge animal body is outer, also secondary pollution and environmental pollution can be caused through drinking-water and food, application number: application number 201220564064.0, the title of utility model: zearalanol detection kit, the technical solution which provides Be: 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 of zearalanol coupled antigen；Reagent bottle includes being equipped with The reagent of the reagent bottle of zearalanol monoclonal antibody solution, sheep anti mouse antiantibody equipped with horseradish peroxidase-labeled Bottle.

At present in the world in vegetable food zearalanol 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, 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.

Utility model content

In order to overcome the shortcomings of prior art, the utility model provides a kind of for detecting the food of zearalanol Detection kit, easy to operate, detection sensitivity is high, at low cost, and it is red can to carry out corn in the multiple samples of one-time continuous progress The measurement of mould alcohol residual quantity reduces the time required for detection sample, can effectively solve the problems in background technique.

The technical scheme adopted by the utility model to solve the technical problem is as follows: a kind of for detecting the food of zearalanol Product detection kit, including kit, the kit bottom are provided with bottom, and the kit is internally provided with installed reagents bottle The hole to match, and the reagent for including in described hole has concentrated cleaning solution, redissolution liquid, antibody working solution, enzyme marker dilute The hole side releasing liquid, terminate liquid, developing solution, confining liquid and titer, and matching is provided with the joint strip of related liquid, the bottom With enzyme frame is set in layer, abacus is set at the top of the enzyme frame, is set with plate rail on the orifice plate, is arranged on the plate rail Show Reagent Tube.

As the utility model a preferred technical solution, the food inspection kit uses sheep anti mouse in the detection Antiantibody, and it is attached with blank ox urine sample.

As the utility model a preferred technical solution, the terminate liquid uses the 2M concentrated sulfuric acid.

Compared with prior art, the utility model has the beneficial effects that

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 reserved for 3 months under the conditions of 2~8 DEG C, the whole system reaction time are as follows: 130 minutes, than instrumental method (gas phase, liquid Phase method etc.) many times are saved, selection compound zearelone, α-corn in structure and as zearalenones Red mould enol, zearalanol, β-zearalenol are reacted as standard items, detect cross reacting rate, kit pair Zearalenone, zearelone, α-zearalenol, the cross reacting rate of zearalanol are higher, can be simultaneously Detection is synchronized to the pure and mild zearalenone of Gibberella zeae, while detecting red mould alcohol, passes through the intersection of zeranol Reactivity maps the concentration of red mould alcohol, so as to fast and accurately detect.

Detailed description of the invention

Fig. 1 is the overall structure diagram of the utility model；

Fig. 2 is the Elisa plate structure schematic diagram of the utility model；

In figure: 1- kit；2- bottom；3- concentrated cleaning solution；4- joint strip；5- antibody working solution；6- developing solution；7- is terminated Liquid；8- titer；9- enzyme frame；10- plate rail；11- Reagent Tube；12- orifice plate；13- redissolves liquid；14- enzyme marker dilution； 15- confining liquid.

Specific embodiment

The following will be combined with the drawings in the embodiments of the present invention, carries out the technical scheme in the embodiment of the utility model Clearly and completely describe, it is clear that the described embodiments are only a part of the embodiments of the utility model, rather than whole Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art are without making creative work Every other embodiment obtained, fall within the protection scope of the utility model.

The explanation of following embodiment be with reference to attached drawing, can be to the particular implementation implemented to example the utility model Example.The direction and position term that the utility model is previously mentioned, for example, "upper", " in ", "lower", "front", "rear", "left", "right", " It is interior ", "outside", " side " etc., be only the direction and position with reference to annexed drawings.Therefore, the direction and position term used is to use To illustrate and understand the utility model, rather than to limit the utility model.

Embodiment:

As depicted in figs. 1 and 2, the utility model provides a kind of for detecting the food inspection reagent of zearalanol Box, including kit 1,1 bottom of kit are provided with bottom 2, and the kit 1 is internally provided with installed reagents bottle and matches Hole, and the reagent for including in described hole have concentrated cleaning solution 3, redissolve liquid 13, antibody working solution 5, enzyme marker dilution Liquid 14, terminate liquid 7, developing solution 6, confining liquid 15 and titer 8, and the hole side to match is provided with the joint strip 4 of related liquid, With enzyme frame 9 is set in the bottom 2, abacus 12 is set at the top of the enzyme frame 9, is set with plate rail on the orifice plate 12 10, Reagent Tube 11 is arranged on the plate rail 10.

Wherein, antigen of the coating antigen of the food inspection kit using zearalanol and ovalbumin coupling, institute The antibody for stating food inspection kit uses the monoclonal antibody of zearalanol and protein coupled antigen, and antigenic dilution is 0.05M carbonate buffer solution, antibody working solution 5 are that 0.05M contains bovine serum albumin and protectant phosphate buffer solution；It is described Confining liquid 15, which contains 0.5% casein, 1% calf serum, the phosphate buffer of 5 ‰ Sodium azides, and its configuration concentration, to be distinguished Calf serum concentration 0.5%, 1%, 2%, 4%, 5% is as five kinds of A, B, C, D, E selections；The enzyme label dilution 14 is small containing 5% Cow's serum and protectant buffer, configuration concentration distinguish calf serum concentration 2%, 4%, 8%, 16%, 30% as A, B, C, D, Five kinds of E selections；The food inspection kit uses sheep anti mouse antiantibody in the detection, and is attached with blank ox urine sample；Institute The B solution for stating solution A and hydrogen peroxide that developing solution 6 is tetramethyl benzidine mixes；The terminate liquid 7 uses the dense sulphur of 2M Acid.

ELISA method of the utility model based on indirect competition, by pre-coated on the capillary strip between adjacent reagent pipe The antigen of zearalanol and ovalbumin coupling, pre-coated corn in the residue zearalanol and capillary strip in sample The antibody of red mould alcohol and the anti-zearalanol of antigenic competition of ovalbumin coupling, is added enzyme marker dilution 14, with colour developing Liquid 6 carries out tmb substrate colour developing, and sample light absorption value and the content of its contained zearalanol are negatively correlated, compared with standard curve Multiplied by the content of its corresponding extension rate you can get it residue zearalanol, the determination 1 of most suitable peridium concentration: 5000, most suitable monoclonal antibody and ELIAS secondary antibody extension rate are determined as 5000:2500, sample solution and monoclonal antibody liquor capacity ratio It is determined as 50:50, coating buffer is determined as B, and confining liquid is determined as D, and ELIAS secondary antibody dilution is determined as D, monoclonal antibody dilution Liquid is determined as D, and substrate developing solution is determined as intermediate concentration；Compbined test is carried out to selected various solution, maximum is inhaled Light value, IC50, blank ox urinate measured value, and 21ng/ml addition measured value meets the requirements.Further experiment can be carried out to determine It obtains: being coated with the 37 DEG C of incubation 1h that are determined as of condition, then 4 DEG C are stayed overnight, off-period is determined as 60min, monoclonal antibody reaction time Be determined as 60min, the ELIAS secondary antibody reaction time is determined as 60min, and substrate color condition is determined as 10min.

Simultaneously in the detection for determining red mould alcohol by the synchronous cross reaction with zearalenone, in the preparation of antigen It is upper: by ZEN and hydroxylamine hydrochloride condensation reaction, to introduce a new hydroxyl, 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 solution~ For 24 hours, revolving removes pyridine and unreacted azanol, obtains crude product, and 5.0ml tetrahydrofuran is added and 200 μ l pyridines, room temperature are stirred It mixes 0.5h, is added dropwise the 50mg succinic anhydride being dissolved in 1.0ml tetrahydrofuran solution, room temperature reaction 12~for 24 hours, revolving removes solvent And pyridine, column chromatographic purifying obtain ZEN haptens.

In the preparation of antibody: after ZEN-BSA is mixed with equivalent freund adjuvant, being immunized in a manner of subcutaneous multi-point injection Balb/c mouse；Mouse boosting cell is won after 4 times immune 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 antibody after purification.

In the preparation of coating antigen: being coupled ZEN haptens and OVA to obtain coating antigen.Concrete operations are as follows: taking ZEN half Antigen 5mg is dissolved with 1.0mlDMF, takes 5 μ l of isobutyl chlorocarbonate to be added, 10 DEG C are stirred to react 30min, and reaction solution can be obtained A；OVA30mg is weighed, is allowed to be substantially dissolved in 2.0ml50mmol/L sodium carbonate liquor 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, saves backup in -20 DEG C.

Double check mode: being diluted to debita spissitudo for coating antigen with coating buffer and be added in ELISA Plate, 1 hole, 100 μ l；37℃ It is incubated for 2h, extraction raffinate is outwelled, is washed 1 time with wash operating solution；1 hole of confining liquid, 150 μ l is added, 37 DEG C of incubation 2h are got rid of wherein molten Liquid pats dry, and 10mg enzyme is dissolved in 0.10mol/LpH6.8PBS of the 0.2ml containing 1.25% glutaraldehyde and is placed at room temperature for 18h, sufficiently thoroughly Analysis removes unreacted glutaraldehyde with SephadexG25 column.Add physiological saline that 1.0ml (containing 5mg) is then added to 1.0ml anti- Liquid solution and 1.0ml1.00mol/LpH9.6 carbonate buffer solution, 4 DEG C of refrigerators are placed for 24 hours after mixing, are added 0.1ml0.20mol/L lysine sets room temperature 2h, is sufficiently dialysed with 0.15mol/LpH7.2PBS liquid, and centrifugation goes to precipitate, supernatant As enzyme conjugates is further applied with ammonium sulfate precipitation after purification, is made of A liquid and B liquid, and A formula of liquid is to take 1g peroxide Change urea, 10.3g citric acid, 35.8gNa2HPO412H2O, 100 μ l Tween-20s are dissolved in deionized water, are adjusted to pH=5.0, and It is finally settled to 1000ml, B formula of liquid is to take 700mg tetramethyl benzidine, and 10.3g citric acid is dissolved in deionized water, is adjusted to pH Value=2.6, and it is finally settled to 1000ml, the sample solution or ZEN of 1 hole, 20 μ l are added into the ELISA Plate for being coated with ZEN antigen Standard solution (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 The enzyme labelled antibody working solution of 1 hole, 100 μ l is added, oscillation reacts 10min in 25 DEG C of light protected environments after mixing.It is clapped after washing 5 times It is dry, substrate A liquid, each 50 μ l in 1 hole of B liquid is added, is terminated after reacting 5min with 1 hole of the concentrated sulfuric acid, the 50 μ l that concentration is 2.00mol/L, and OD value (OD) is detected with microplate reader, standard curve, selection are drawn according to the logarithm of percentage absorbance value and standard concentration Compound zearelone, α-zearalenol, zearalanol, β-corn in structure and as zearalenones Red mould enol is reacted as standard items, detects cross reacting rate.

In conclusion the utility model is mainly characterized by: kit detection is limited to 0.5ng/ml, TIANZHU XINGNAO Capsul Between 70.0%~116.0%, the coefficient of variation can be reserved for 3 months under the conditions of 2~8 DEG C less than 20%, whole system reaction Time are as follows: 130 minutes, save many times than instrumental method (gas phase, liquid phase process etc.), selection is in structure and Gibberella zeae Compound zearelone, α-zearalenol, zearalanol, β-zearalenol are as standard items as ketene It is reacted, cross reacting rate is detected, the results show that kit is to zearalenone, zearelone, α-Gibberella zeae alkene Alcohol, the cross reacting rate of zearalanol are higher, can synchronize inspection to the pure and mild zearalenone of Gibberella zeae simultaneously It surveys, while detecting red mould alcohol, 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 present invention is not limited to the details of the above exemplary embodiments, and And without departing substantially from the spirit or essential attributes of the utility model, it can realize that this is practical new in other specific forms Type.Therefore, in all respects, the present embodiments are to be considered as illustrative and not restrictive, this is practical new The range of type is indicated by the appended claims rather than the foregoing description, it is intended that containing for the equivalent requirements of the claims will be fallen in All changes in justice and range are embraced therein.It should not treat any reference in the claims as limiting Related claim.

Claims (3)

1. a kind of for detecting the food inspection kit of zearalanol, it is characterised in that: including kit (1), the examination Agent box (1) bottom is provided with bottom (2), and the kit (1) is internally provided with the hole that installed reagents bottle matches, and the hole The reagent for including in hole has concentrated cleaning solution (3), redissolves liquid (13), antibody working solution (5), enzyme marker dilution (14), end Only liquid (7), developing solution (6), confining liquid (15) and titer (8), and the hole side to match is provided with the joint strip of related liquid (4), abacus (12) are set at the top of the enzyme frame (9), the orifice plate with being set with enzyme frame (9) in the bottom (2) (12) it is set on plate rail (10), is arranged with Reagent Tube (11) on the plate rail (10).

2. according to claim 1 a kind of for detecting the food inspection kit of zearalanol, it is characterised in that: institute It states food inspection kit and uses sheep anti mouse antiantibody in the detection, and be attached with blank ox urine sample.

3. according to claim 1 a kind of for detecting the food inspection kit of zearalanol, it is characterised in that: institute Terminate liquid (7) are stated using the 2M concentrated sulfuric acid.