CN109752524A - The enzyme linked immunological kit of diethylstilbestrol and its application in a kind of detection feed - Google Patents
The enzyme linked immunological kit of diethylstilbestrol and its application in a kind of detection feed Download PDFInfo
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- CN109752524A CN109752524A CN201711057842.0A CN201711057842A CN109752524A CN 109752524 A CN109752524 A CN 109752524A CN 201711057842 A CN201711057842 A CN 201711057842A CN 109752524 A CN109752524 A CN 109752524A
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Abstract
The present invention provides a kind of enzyme linked immunological kits for detecting diethylstilbestrol, it contains: being coated with the ELISA Plate diethylstilbestrol standard solution of diethylstilbestrol coupled antigen, enzyme conjugates, enzyme conjugates working solution, substrate developing solution, terminate liquid is concentrated.The invention also discloses a kind of methods using diethylstilbestrol in above-mentioned enzyme linked immunological kit detection sample, it includes: to carry out Sample pretreatment first, is then detected with kit, ultimate analysis testing result.Enzyme linked immunological kit provided by the invention can be used for detecting the residual quantity of diethylstilbestrol in feed (raw material, batch and concentrate feed) sample, and easy to operate, at low cost, high sensitivity on-site supervision and can be suitble to the screening of great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technologies, and in particular to a kind of to exempt from for detecting the enzyme-linked of diethylstilbestrol in feed
Epidemic disease kit, it is suitable for diethylstilbestrols in feed (raw material, batch, concentrate feed) to measure.
Background technique
Diethylstilbestrol (Diethylstilbestrolum, DES) molecular formula is C18H20O2, it is a kind of artificial synthesized female
The immunity of livestock and poultry can be improved in hormone, shortens the growth cycle of livestock and poultry, cheap because its effect is obvious, is often used in
The fowl poultry kinds animal such as pig, ox, sheep, chicken, duck, in the cultivation of the aquatic products such as fish, shrimp, people be eaten for a long time this " having anti-livestock and poultry ",
" having anti-fishes and shrimps " etc. can cause to accumulate in human body, and it is bad that human body excess intake will appear Nausea and vomiting, anorexia, headache etc.
Reaction, prolonged application can induce reproductive system malignant tumour, and women long-time service can be such that endometrial hyperplasia excessively leads
Uterine hemorrhage and metrauxe, pregnant woman is caused to will lead to fetal congenital defect.To ensure animal food safety, maintenance
People's health, the Ministry of Agriculture in 2002 have issued No. 176 bulletins, have disclosed and forbid in feed, animal drinking water and livestock and poultry water
It produces and uses DES in cultivation, but due to using DES remarkable in economical benefits, illegal abuse phenomenon is still had.
Ke Biao chemical analysis inspection center is referring to No. 1163 bulletin -9- of national standard GB/T5009.108-2003 and the Ministry of Agriculture
2009 have carried out method confirmation using high performance liquid chromatography, obtain good result, and this method can detecte livestock meat, aquatic products
DES content in middle DES residual quantity and feed.Ke Biao chemical analysis inspection center has passed through China national certification and accreditation supervision pipe
Two-in-one (the CMA, CNAS) laboratory certification of the reason committee and China National Accreditation Service for Conformity Assessment is approved, power can be provided
Third party's examining report of prestige.
Summary of the invention
The purpose of the present invention is to provide a kind of enzyme linked immunological kit for diethylstilbestrol assay, operation letters
It is single, it is suitble to the screening of scene batch samples.
Kit of the present invention, it includes:
(1) it is coated with the ELISA Plate of diethylstilbestrol coupled antigen；
(2) diethylstilbestrol standard solution；
(3) enzyme conjugates is concentrated；
(4) enzyme combination diluent；
(5) substrate developing solution；
(6) terminate liquid.
The diethylstilbestrol coupled antigen is the conjugate of diethylstilbestrol haptens and carrier protein, and the carrier protein can
For ovalbumin, bovine serum albumin(BSA), hemocyanin, thyroprotein, mouse haemocyanin, human albumin.
The diethylstilbestrol haptens preparation process:
1) 20mg diethylstilbestrol is taken, the 4-dimethylaminopyridine of 10ul, 1,3- propane diamine, catalytic amount are dissolved in 2ml
N in N '-dimethyl formamide, obtains I liquid；
2) 20mg N is taken, N '-dicyclohexylcarbodiimide is dissolved in 0.5mlDMF, obtains II liquid；
3) under the conditions of 0 DEG C, II liquid is slowly added dropwise into I liquid, after restoring room temperature, the reaction was continued 20 h；
4) solvent is evaporated off, column chromatographs (eluent: methylene chloride/methanol, volume ratio 20:1), obtains diethylstilbestrol haptens.
The concentration enzyme conjugates is the diethylstilbestrol monoclonal antibody of enzyme label, and the marker enzyme is horseradish peroxidase
Enzyme；Marker enzyme is coupled to obtain by enzyme conjugates using Over-voltage protection with antibody.
In order to be more convenient on-site supervision and great amount of samples screening, the kit further include diethylstilbestrol standard solution,
Enzyme combination diluent, substrate developing solution, terminate liquid.
The terminate liquid is the sulfuric acid or hydrochloric acid solution of 1 ~ 2mol/L, and the substrate developing solution is by substrate solution A liquid and bottom
Thing liquid B liquid composition, substrate solution A liquid are urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
The enzyme combination diluent is PH7.2 ~ 7.4,0.5% ~ 1% bovine serum albumin(BSA), the phosphate of 0.1mol/L
Buffer, the percentage are weight percentage.
Wherein ELISA Plate make it is each used in the process of coating buffer be pH9.6,0.1mol/L carbonate buffer solution,
Confining liquid used is PH7.2 ~ 7.4,0.5% ~ 1% bovine serum albumin(BSA), 0.1mol/L phosphate buffer, the percentage
It is weight percentage.
The preparation process of ELISA Plate in the present invention are as follows: coating antigen is diluted to 0.1 ~ 0.2ug/ml with coating buffer, often
100 ul are added in hole, and 37 DEG C incubate 2h or 4 DEG C overnight, and coating buffer of inclining is washed 2 times with cleaning solution, and each 30s is patted dry, then
150 ~ 200ul confining liquid is added in every hole, 37 DEG C of incubations l ~ 2h, liquid pats dry in hole of inclining, and uses aluminium film vacuum close after dry
Envelope saves.
Testing principle of the invention are as follows:
When diethylstilbestrol coupled antigen pre-coated on capillary strip remains in sample after sample solution or standard solution is added
Diethylstilbestrol and ELISA Plate on pre-coated diethylstilbestrol coupled antigen competition diethylstilbestrol enzyme conjugates, developed the color with substrate
Liquid colour developing, in sample light absorption value and the content of contained diethylstilbestrol are negatively correlated, and you can get it compared with standard curve sample oneself
The content of the female phenol of alkene.Simultaneously according to the depth of color on ELISA Plate, with the diethylstilbestrol standard solution color of series of concentrations
Compare can in rough judgement sample diethylstilbestrol concentration range.
The present invention also provides a kind of method using above-mentioned enzyme linked immunological kit diethylstilbestrol, it comprising steps of
(1) sample pre-treatments；
(2) it is detected with kit；
(3) analysis detection result.
The enzyme linked immunological kit that the present invention detects diethylstilbestrol mainly uses hexene in competitive ELISA method test sample
The content of female phenol；Low to the pre-treatment requirement of sample, sample pretreatment process is simple, quickly can detect batch samples simultaneously,
Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, have specific height, high sensitivity, accuracy high, smart
The features such as exactness is high.Enzyme linked immunological kit of the invention, carrying convenience, easy to use, structure is simple, detection method is quick,
Simplicity is suitable for batch samples screening.
Detailed description of the invention
The canonical plotting of Fig. 1 kit.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to model a side gate of an imperial palace of the invention.
The preparation of 1 reagent constituents of embodiment
1, diethylstilbestrol haptens system is each
1) 20mg diethylstilbestrol is taken, 10ul, 1,3- propane diamine, the 4-dimethylaminopyridine of catalytic amount is dissolved in 2mlN, N '-diformazan
In base formamide, I liquid is obtained；
2) 20mg N is taken, N '-dicyclohexylcarbodiimide is dissolved in 0.5mlDMF, obtains II liquid；
3) under the conditions of 0 DEG C, II liquid is slowly added dropwise into I liquid, after restoring room temperature, the reaction was continued 20 h；
4) solvent is evaporated off, column chromatographs (eluent: methylene chloride/methanol, volume ratio 20:1), obtains diethylstilbestrol haptens, such as
Fig. 1.
2, the preparation of antigen
Immunogene preparation --- diethylstilbestrol haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
1) it takes diethylstilbestrol haptens 5.3mg 0.5ml DMF to dissolve, obtains I liquid；
2) it takes BSA 30mg 2.0ml, pH7.0,0.1mol/L phosphate buffer to dissolve, obtains II liquid；
3) carbonization two industry amine (EDC) 10mg 0.5ml, pH7.0,0.1mol/L phosphate buffers dissolution is taken, III liquid is obtained:
4) I liquid is mixed with Il liquid, is slowly added dropwise under stiring into III liquid, reacted at room temperature 2 hours, 4 DEG C are dialysed two days, daily
It changes liquid three times, obtains immunogene.
Coating antigen preparation --- diethylstilbestrol haptens and ovalbumin (OVA) coupling obtain coating antigen.
1) it takes diethylstilbestrol haptens 5.3mg 0.5ml DMF to dissolve, obtains I liquid；
2) it takes OVA l8mg 2.0ml, pH7.0,0.1mol/L phosphate buffer to dissolve, obtains II liquid；
3) EDC10mg 0.5ml, pH7.0 are taken, the dissolution of 0.1mol/L phosphate buffer obtains III liquid:
4) I liquid is mixed with II liquid, is slowly added dropwise under stiring into III liquid, the mixed reaction in room 2 hours, 4 DEG C are dialysed two days, daily
It changes liquid three times, obtains coating antigen.
3, the preparation of diethylstilbestrol monoclonal antibody
A. animal immune
The immunizing antigen that above-mentioned steps are obtained is injected into Balb/c Mice Body, and immunizing agent l50ug/ the most only, makes its generation
Antiserum.
B. cell fusion and cloning
Immune Bal b/c mouse boosting cell is taken, screening is merged in 9:1 (number most matches) ratio with SP2/0 myeloma cell and is obtained
The diethylstilbestrol monoclonal antibody hybridoma cell strain of the reddish brown diethylstilbestrol monoclonal antibody of stably excreting.
C. cell cryopreservation and recovery
L × 10 is made with frozen stock solution in hybridoma9The cell suspension of a/ml, saves for a long time in liquid nitrogen.It is taken when recovery
Cryopreservation tube out is immediately placed in 37 DEG C of water-bath middling speeds and melts, and after centrifugation removal frozen stock solution, moves into culture culture in glassware.
D. the system of monoclonal antibody respectively with purifying
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, is saturated with octanoic acid one
Ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
4, the preparation of ELISA Plate
Coating antigen is diluted to 0.1 ~ 0.2ug/ml with coating buffer, 100 u l, 2h or 4 DEG C of mistake of 37 DEG C of incubations is added in every hole
Night, coating buffer of inclining are washed 2 times with cleaning solution, and each 30s is patted dry, and 50 ~ 200 u l of l closing is then added in every hole
Liquid, 37 DEG C of incubation l ~ 2h, liquid pats dry in hole of inclining, and is saved after dry with aluminium film vacuum sealing.
5, the preparation of enzymic-labelled antibody
Antibody and horseradish peroxidase (HRP) are subjected to coupling using Over-voltage protection and prepare enzymic-labelled antibody, enzyme and antibody
Molar concentration rate be 2:1.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of diethylstilbestrol
The enzyme linked immunological kit for setting up detection diethylstilbestrol, makes that it includes following components:
(1) it is coated with the ELISA Plate of diethylstilbestrol coupled antigen；
(2) 6 bottles of diethylstilbestrol standard solution, concentration are respectively 0ug/L, 1ug/L, 3ug/L, 9ug/L, 27ug/L, 81ug/
L。
(3) enzyme conjugates is concentrated；
(4) enzyme combination diluent；
(5) substrate developing solution is made of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide, and substrate solution B liquid is four
Methyl biphenyl amine；
(6) terminate liquid is 2mol/L sulfuric acid.
The detection of diethylstilbestrol in 3 sample of embodiment
1. sample pre-treatments
With homogenizer homogeneous feed sample；Feed sample as low as 50ml polystyrene centrifuge tube after weighing 5.0g ± 0.05g homogeneous
In, 50% methanol of 25m1 is added, acutely vibrates 5min, 3000r or more, room temperature (20-25 DEG C/68-77 ℉) centrifugation with oscillator
5min；It takes 500 u l supernatants into 2ml polystyrene centrifuge tube, 500 ul10% sodium-chloride water solution oscillators is added
1min is vibrated, is mixed；Take 20 u l for analyzing.
2. being detected with kit
Ochratoxin A standard solution/20 u of sample is added into the micropore of enzyme marker plate for being coated with diethylstilbestrol coupled antigen
L, add 100 u 1 of enzyme conjugates working solution (by be concentrated enzyme conjugates enzyme combination diluent according to l:20 volume into
Row dilution), with cover board film sealing plate, 25 DEG C are protected from light 10min, pour out liquid in hole, and every hole is added 250 u l deionized waters and fills
Divide washing 4-5 times, every minor tick 10s is patted dry with blotting paper, and 50 u l of substrate solution A liquid urea peroxide, substrate solution B is added in every hole
50 u l of liquid tetramethyl benzidine (TMB), gently oscillation mixes, and 25.DEG C insulating box is protected from light colour developing 5min, and 2mol/L is added in every hole
50 u l of terminate liquid sulfuric acid, gently oscillation mixes, and is set at 450nm with microplate reader wavelength, measures every hole absorbance value (OD
Value).
3. Analysis of test results
With the absorbance values (B) of the standard solution of each concentration obtained divided by first standard solution (0 mark
It is quasi-) absorbance value (Bo) multiplied by 100%, obtain percentage absorbance value.With pair of diethylstilbestrol standard concentration (ug/L)
Numerical value is X-axis, and percentage absorbance value is Y-axis, draws canonical plotting.The percentage extinction of sample solution is calculated with same method
Angle value, the concentration of each corresponding sample can then read the content of diethylstilbestrol from standard curve.
4 diethylstilbestrol enzyme linked immunological kit sensitivity of embodiment, preci-sion and accuracy, storage life experiment
1. kit sensitivity and sword detection limit
Conventionally assay kit sensitivity test, kit sensitivity is 1ug/L, respectively to 20 parts of blank feeds
(raw material, batch, concentrate feed) sample is detected, and the concentration corresponding to each percentage absorptance is found from standard curve, with
The average value of 20 parts of sample diethylstilbestrol concentration indicates detection limit plus 3 times of standard deviations, as a result this method to feed (raw material,
Batch, concentrate feed) pattern detection is limited to 0 ug/kg of l.
2. kit accuracy and precision
Using the testing result coefficient of variation (CV%) of a certain concentration samples of replication as precision evaluation index.With the rate of recovery
As accuracy estimating index.Coefficient of variation CV% calculation formula are as follows: CV%=SD/X × l00%；Wherein SD is standard deviation, X
For the average value of determination data.Rate of recovery calculation formula are as follows: the rate of recovery (%)=actual measured value/theoretical value × l00%.Wherein
Theoretical value is the addition concentration of analog sample.
By 10ug/kg, 20ug/kg, 40 ug/kg, tri- concentration diethylstilbestrols to raw material, batch, concentrate feed sample into
Row addition recycling measurement, each sample do 4 in parallel, are measured with three batches of different reagents, calculate the average recovery rate of sample
And precision result see the table below 1.
1 precision of table and accuracy test
Feed (raw material, batch, concentrate feed) sample is added with l0,20, the diethylstilbestrol of tri- concentration of 40ug/kg,
Average recovery rate is between 87.8% ~ l02.8%；The coefficient of variation is respectively less than 20%.The preci-sion and accuracy of testing result accords with
Close relevant criterion requirement.
3. kit storage life is tested
Kit preservation condition is 2 ~ 8 DEG C, by measurement in l2 months, the maximum absorbance value (zero standard) of kit, 50%
Inhibition concentration, diethylstilbestrol addition actual measured value are within normal range (NR).Consider to have non-in transport and use process
Normal preservation condition occurs, and kit is placed 7 days under 37 DEG C of preservation conditions, carries out accelerated aging tests, the results showed that should
Kit indices comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C of refrigerator freezings
7 days, measurement result also indicated that kit indices are completely normal.It can show that kit can be protected at 2 ~ 8 DEG C from result above
It deposits l2 months.
Claims (10)
1. a kind of enzyme-linked immunologic detecting kit for detecting diethylstilbestrol, it is characterised in that include:
(1) it is coated with the ELISA Plate of diethylstilbestrol coupled antigen；
(2) diethylstilbestrol standard solution；
(3) enzyme conjugates is concentrated；
(4) enzyme combination diluent；
(5) substrate developing solution；
(6) terminate liquid.
2. kit as claimed in claim l, it is characterised in that the diethylstilbestrol coupled antigen is anti-by diethylstilbestrol half
Original is obtained with carrier protein couplet, and the diethylstilbestrol haptens structure is as follows:
。
3. kit as claimed in claim 2, it is characterised in that the preparation method of the diethylstilbestrol haptens is main
Include the following steps:
1) 20 mg diethylstilbestrols are taken, the 4-dimethylaminopyridine of 10ul, 1,3- propane diamine, catalytic amount are dissolved in 2ml
N in N '-dimethyl formamide, obtains I liquid；
2) 20mg N is taken, N '-dicyclohexylcarbodiimide is dissolved in 0.5mlDMF, obtains II liquid；
3) under the conditions of 0 DEG C, II liquid is slowly added dropwise into I liquid, after restoring room temperature, the reaction was continued 20 h；
4) solvent is evaporated off, column chromatographs (eluent: methylene chloride/methanol, volume ratio 20:1), and it is anti-to obtain diethylstilbestrol half
It is former.
4. kit as claimed in claim l, it is characterised in that the concentration enzyme conjugates is the diethylstilbestrol of enzyme label
Monoclonal antibody.
5. kit as claimed in claim l, it is characterised in that the marker enzyme of the concentration enzyme conjugates is horseradish peroxidating
Object enzyme；Enzyme conjugates is to be obtained enzyme marker and antibody coupling using Over-voltage protection.
6. kit as claimed in claim l, it is characterised in that: the terminate liquid is the sulfuric acid or hydrochloric acid of 1 ~ 2mol/L
Solution, the substrate developing solution are made of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide solution, bottom
Thing liquid B liquid is tetramethyl biphenyl amine aqueous solution.
7. kit as claimed in claim l, it is characterised in that coating buffer used is PH9.6,0.1mol/L carbonate
Buffer, confining liquid used be PH7.2 ~ 7.4,0.5% ~ 1% bovine serum albumin(BSA), 0.1mol/L phosphate buffer, described hundred
Ratio is divided to be weight percentage.
8. kit as claimed in claim l, it is characterised in that the enzyme combination diluent is PH7.2 ~ 7.4, is contained
0.5% ~ 1% bovine serum albumin(BSA), the phosphate buffer of 0.1mol/L, the percentage are attached most importance to most percentage.
9. kit as claimed in claim l, feature diethylstilbestrol standard solution concentration described in r be respectively 0ug/L,
1ug/L、3ug/L、9ug/L、27ug/L、81ug/L。
10. the method for diethylstilbestrol content, key step include: in a kind of test sample
1) sample to be tested is subjected to pre-treatment；
2) enzyme linked immunological kit described in claim 1-9 any one is detected；
3) analysis detection result.
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| Application Number | Priority Date | Filing Date | Title |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711057842.0A CN109752524A (en) | 2017-11-01 | 2017-11-01 | The enzyme linked immunological kit of diethylstilbestrol and its application in a kind of detection feed |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766633A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting stilbestrol residue and detection method thereof |
| CN101059511A (en) * | 2006-05-18 | 2007-10-24 | 李季 | ELISA reagent kit suitable for diethylstilbestrol residue analysis |
| CN105572363A (en) * | 2014-10-11 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | ELISA kit for detecting roxarsone, and application thereof |
-
2017
- 2017-11-01 CN CN201711057842.0A patent/CN109752524A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766633A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting stilbestrol residue and detection method thereof |
| CN101059511A (en) * | 2006-05-18 | 2007-10-24 | 李季 | ELISA reagent kit suitable for diethylstilbestrol residue analysis |
| CN105572363A (en) * | 2014-10-11 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | ELISA kit for detecting roxarsone, and application thereof |
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