CN1563993A - Enzyme-linked immune kit for detecting corn bakanae alcohol - Google Patents
Enzyme-linked immune kit for detecting corn bakanae alcohol Download PDFInfo
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- CN1563993A CN1563993A CN 200410029806 CN200410029806A CN1563993A CN 1563993 A CN1563993 A CN 1563993A CN 200410029806 CN200410029806 CN 200410029806 CN 200410029806 A CN200410029806 A CN 200410029806A CN 1563993 A CN1563993 A CN 1563993A
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Abstract
Disclosed kit includes specificity antibody of corn zeranol, coated antibody/antigen and enzyme labeled corn zeranol. The disclosed kit is capable of testing large quantities of samples rapidly, and reagent is supplied in form of treatment fluid. Features are: easy of use, high specificity, sensitivity, accuracy and low cost. The kit can play important function in detecting residual of corn zeranol in animal foodstuff.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects ZER in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
(zeranol ZER), belongs to mine-mooring cable acid lactone non-steroid class anabolic hormone to ZER.ZER is as the cattle and sheep growth accelerator, can promote the synthetic of protein, can improve carcass lean meat percentage and feed conversion rate, but it has weak estrogen action, residual meeting in animal tissue causes the human body disorder and influences the normal development of secondary sex characters, externally condition is induced down, also may be carcinogenic.After ZER discharges outside the animal body, also can cause secondary pollution and environmental pollution through drinking-water and food.European Union and China Ministry of Agriculture clearly forbid hormone medicines such as ZER are applied to livestock and poultry cultivation.In Dec, 2002, China Ministry of Agriculture announced the literary composition regulation No. 235, and it is residual to detect ZER in all edible tissues of all food animals.But because ZER is effective as the cattle and sheep loading agent, the economy return height, illegal use is still very general.Therefore strengthen the residue detection of ZER in the animal food is very important.
At present, the main residual quantity that adopts instrumental method to detect ZER, as thin-layered chromatography (TLC), vapor-phase chromatography (GC), high pressure lipuid chromatography (HPLC) (HPLC), gas-matter online (GC/MS), liquid-matter online (HPLC/MS), Capillary Electrophoresis (CE) etc., because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.
The innovation and creation content
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects ZER.
The enzyme linked immunological kit of detection ZER provided by the present invention comprises that two of ZER specific antibody, bag quilt resists and enzyme mark ZER.
Wherein, described ZER specific antibody can be ZER monoclonal antibody or ZER polyclonal antibody; Described ZER monoclonal antibody or polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source and cavy source antibody, described ZER monoclonal antibody is preferably the ZER mouse monoclonal antibody, and described ZER polyclonal antibody is preferably the ZER rabbit polyclonal antibody.Above antibody all can prepare as immunogene according to a conventional method with the conjugate of ZER and carrier protein.Described carrier protein can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), ovalbumin (OVA) or hemocyanin common carrier albumen such as (KLH); The conjugate of described ZER and carrier protein can obtain by the pure and mild carrier protein of Gibberella zeae is carried out coupling with mixed anhydride method.Described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase.Horseradish peroxidase can adopt several different methods of the prior art such as glutaraldehyde method, and the periodates method is crosslinked on ZER, wherein is preferably glutaraldehyde method.Described two anti-anti-mouse or the anti-rabbit antiantibodys of can be.
The material that can be used as fixing described two carriers that resist is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises ZER standard solution, developer, stop buffer, concentrated cleaning solution and redissolution liquid.
Described concentrated cleaning solution is the phosphate buffer that contains the 0.8%-1.2% tween; Described developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, and described colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine (OPD) or tetramethyl benzidine (TMB); Described redissolution liquid is the phosphate buffer that contains 2% methyl alcohol.
Detection principle of the present invention is adsorbed on the solid phase carrier for resisting two, add sample and enzyme mark ZER, add the ZER specific antibody again, the pure and mild enzyme mark of Gibberella zeae ZER residual in the testing sample is competed specific antibody, the colour developing back stops, the working sample light absorption value, the ZER residual quantity is negative correlation in this value and the sample, relatively can draw the content of ZER with typical curve.Simultaneously according to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the ZER standard solution color of series concentration.
The enzyme linked immunological kit of detection ZER of the present invention mainly adopts the residual quantity of ZER in the qualitative or samples such as detection by quantitative animal tissue, urine sample of indirect competitive ELISA method; This kit cost is low, and low to the pre-treatment requirement of sample, sample pretreatment process is simple, simultaneously the fast detecting gross sample; This kit adopts the ZER monoclonal antibody or the polyclonal antibody of high specific, main agents all provides with the working fluid form, easy to use, have characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can in animal food ZER residue detection, play a significant role.
Description of drawings
Fig. 1 is the structural representation of the enzyme linked immunological kit of detection ZER
Embodiment
The preparation of embodiment 1, antigen and antibody
(1) immunogenic synthetic
Adopt mixed anhydride method to carry out coupling the pure and mild bovine serum albumin(BSA) of Gibberella zeae (BSA) and obtain immunogene.
(2) ZER mouse monoclonal antibody preparation
The animal immune program adopts BALB/c mouse as immune animal, with ZER and bovine serum albumin(BSA) conjugate is immunogene, immunizing dose is 80-100 μ g/, Freund's complete adjuvant with antigen and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
Immune BALB/c mouse splenocyte is got in Fusion of Cells and cloning, and in 5-10: 1 ratio and SP2/0 myeloma cell are merged, and adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
Cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6-5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, and BALB/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 7-14 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6Individual/as only, to gather ascites after 7-10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
(3) preparation of ZER rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with ZER and bovine serum albumin(BSA) conjugate is immunogene, immunizing dose is 1mg/kgb.w., Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3-4 week adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Take a blood sample behind the last immune 7-10d, measure serum antibody titer, the arteria carotis bloodletting obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
(4) two anti-preparations
As immune animal, is that immunogene carry out immunity with mouse or rabbit igg with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody.
The enzyme linked immunological kit of embodiment 2, detection ZER
(1) structure of the enzyme linked immunological kit of detection ZER
The structure of kit as shown in Figure 1, mainly by box body 1, vacuum-packed 96 holes of aluminium film/2,6 bottles of ZER series concentration standard items 3 of 40 hole ELISA Plate, enzyme labeling thing working fluid 4, ZER antibody working fluid 5, substrate colour developing liquid A liquid 6, substrate colour developing liquid B liquid 7, stop buffer 8, concentrated cleaning solution 9, redissolution liquid 10 and foam carriage 11, be shaped on shrinkage pool on the foam carriage 11, mentioned reagent bottle 3-10 is placed in the shrinkage pool of foam carriage, and foam carriage 11 and ELISA Plate 2 are placed in the box body.Wherein ELISA Plate 2 is made up of plastic stent and detachable plastic strip.
(2) preparation of agents useful for same
A. ZER standard solution: 6 bottles of ZER series standard solution, 1-3ml/ bottle.
B. bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.05mol/L.
C. confining liquid: 3-10% calf serum.
D. concentrated cleaning solution: contain the phosphate buffer (0.01M pH7.4) of 0.8%-1.2% tween, for the 15-25 of normal working concentration doubly, 30-50ml/ bottle, 1 bottle.
E. ZER antibody working fluid: it is that 0.5-5.0 μ g/L uses 5-8ml/ bottle, 1 bottle that antibody dilution is become protein concentration.
F. enzyme labeling thing working fluid: enzyme mark ZER working fluid, 5-8ml/ bottle, 1 bottle.
G. substrate colour developing liquid A liquid: hydrogen peroxide or urea peroxide, 5-8ml/ bottle, 1 bottle.
H. substrate colour developing liquid B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB), 5-8ml/ bottle, 1 bottle.
I. stop buffer: 1-2mol/L sulfuric acid or hydrochloric acid, 5-8ml/ bottle, 1 bottle.
J. redissolve liquid: contain the phosphate buffer of 2% methyl alcohol, 30-50ml/ bottle, 1 bottle.
(3) preparation of ELISA Plate
Being cushioned liquid with bag will resist rabbit or anti-mouse-anti antibody dilution to become 0.1-1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ are spent the night, coating buffer inclines, with cleansing solution washing 3 times, each 30 seconds, pat dry, in every hole, add 150-200 μ l confining liquid then, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
The pre-treatment of embodiment 3, sample
(1) animal tissue takes by weighing the sample after the 5g homogenate, adds 10ml sodium-acetate buffer and 25 μ l GRD beta-glucuronidase, whirling motion 1min, and 37 ℃ are spent the night then.Add the 8ml ether, behind the whirling motion 3min, the centrifugal 10min of 3000rpm.Repeat once.Ether is merged mutually, and 40 ℃ of nitrogen dry up.Residue is dissolved in the 1ml chloroform, with 2.5ml1M NaOH solution extraction twice, combining extraction liquid, and behind the whirling motion 1min, the centrifugal 10min of 3000rpm.Keep water, add 0.5ml 12M phosphate buffer, use C then
18The post purifying obtains testing sample solution.
(2) urine sample is got the 0.5ml urine, adds 3ml 50mM sodium-acetate buffer, and pH4.8 adds 8 μ l GRD beta-glucuronidase, hatches 3h for 37 ℃, uses C then
18The post purifying obtains testing sample solution.
Residual ZER in embodiment 4, the detection ox urine samples
(1) get proper amount of fresh or thaw after for the examination urine, the centrifugal 10min of 6000rpm gets supernatant and crosses 0.45 μ m filter membrane.Get the sodium-acetate buffer that 500 μ l urines add 3ml 50mM pH4.8.Add 8 μ l β-Pu Taotanggansuanmei/sulfatases, whirling motion 30s leaves standstill 3h in 37 ℃ of constant temperature ovens.Use C18 post purifying then.
(2) with 3ml 100% methyl alcohol activation C18 post, flow speed control is at 1 droplet/second.Sodium-acetate buffer drip washing C18 post with 2ml 50mM pH4.8.Add the urine sample of 500 μ l after step (1) is extracted.Sodium-acetate buffer washing C18 post with 2ml 50mM pH4.8.With the methanol solution washing C18 post of 3ml 40%, positive pressure blowing.With the methanol solution wash-out of 1ml 80% and collect drip, the C18 post after positive pressure blowing drips to the greatest extent is to collect fully.60 ℃ of following nitrogen dry up, and add 500 μ l redissolution liquid in the receiving flask after drying up, and whirling motion 30s as sample solution, measures for the ELISA method.
(3) sample detection
In 96 hole ELISA Plate micropores, add series standard solution or sample solution 50 μ l with sheep anti mouse antiantibody bag quilt, add ZER mouse monoclonal antibody working fluid 50 μ l then, add horseradish peroxidase-labeled ZER working fluid 50 μ l again, with cover plate film shrouding, react 1h in 37 ℃ of constant temperature ovens.Pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole after 30 seconds, pats dry with thieving paper, and so repetitive operation is washed plate 5 times altogether.Every hole adds substrate colour developing liquid A liquid 50 μ l, adds B liquid 50 μ l again, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15-30min.Every hole adds stop buffer 50 μ l, and the mixing that vibrates is gently used microplate reader, measures every hole absorbance (OD value).
The used reagent of present embodiment is all prepared according to the reagent compound method among the embodiment 2.
Each the concentration standard solution that is obtained and the mean value (B) of sample absorbance are divided by the absorbance (B of first standard (0 standard)
0) multiply by 100 again, i.e. percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.Natural logarithm value with Gibberella zeae determining alcohol (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map, and the concentration of corresponding each sample (μ g/L) can be read from typical curve.Also can use regression equation method, calculate sample solution concentration.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.Whole testing process only needed just can finish in 2.5 hours, and lowest detection is limited to 0.3 μ g/L.
(1) kit precision test
The standard repeatability
From three batches of elisa plates, every plate is extracted 20 micropores out, measures same concentration standard solution absorbency value (OD value), and replication 10 times calculates the coefficient of variation.The result shows that coefficient of variation scope is at 8-15%.
The sample repeatability
Get the ZER standard specimen of variable concentrations, add in the sample, get each three of the kits of three different batches respectively, each concentration repeats 5 times.Calculate respectively in the plate, in batch, interassay coefficient of variation.The result shows that the Variation Lines number average of beef is lower than 18%, and the Variation Lines number average of mutton is lower than 18%, and the Variation Lines number average of ox urine sample and sheep urine sample is lower than 15%.
(2) accuracy of kit
Get the ZER standard specimen of 4 concentration, sample added recovery test, each concentration establish 4 parallel, calculate recovery rate respectively.The result shows that beef is 40-120%, and mutton is 40-120%, and the ox urine sample is 60-110%, and the sheep urine sample is 60-110%.
(3) kit storage life test
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, ZER were added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
Claims (9)
1, a kind of enzyme linked immunological kit that detects ZER comprises that two of ZER specific antibody and bag quilt resists and enzyme mark ZER.
2, enzyme linked immunological kit according to claim 1 is characterized in that: described kit also comprises ZER standard solution, developer, concentrated cleaning solution, stop buffer and redissolution liquid.
3, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described ZER specific antibody is ZER monoclonal antibody or ZER polyclonal antibody; They all are that conjugate with ZER and carrier protein obtains as immunogene.
4, enzyme linked immunological kit according to claim 3 is characterized in that: described carrier protein is bovine serum albumin(BSA), human serum albumins, ovalbumin or hemocyanin.
5, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described marker enzyme is horseradish peroxidase or alkaline phosphatase.
6, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described two anti-are anti-mouse or anti-rabbit antiantibody.
7, enzyme linked immunological kit according to claim 2 is characterized in that: described concentrated cleaning solution is the phosphate buffer that contains the 0.8%-1.2% tween.
8, enzyme linked immunological kit according to claim 2 is characterized in that: described developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, and described colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine.
9, enzyme linked immunological kit according to claim 2 is characterized in that: described redissolution liquid is the phosphate buffer that contains 2% methyl alcohol.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410029806 CN1239909C (en) | 2004-03-25 | 2004-03-25 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410029806 CN1239909C (en) | 2004-03-25 | 2004-03-25 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
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| Publication Number | Publication Date |
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| CN1563993A true CN1563993A (en) | 2005-01-12 |
| CN1239909C CN1239909C (en) | 2006-02-01 |
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| CN 200410029806 Expired - Fee Related CN1239909C (en) | 2004-03-25 | 2004-03-25 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107831320A (en) * | 2017-12-13 | 2018-03-23 | 百奥森（江苏）食品安全科技有限公司 | A kind of food inspection kit for being used to detect ZER |
-
2004
- 2004-03-25 CN CN 200410029806 patent/CN1239909C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107831320A (en) * | 2017-12-13 | 2018-03-23 | 百奥森（江苏）食品安全科技有限公司 | A kind of food inspection kit for being used to detect ZER |
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| Publication number | Publication date |
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| CN1239909C (en) | 2006-02-01 |
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