CN109061168A - Detect enzyme linked immunological kit and its application of diclazuril - Google Patents
Detect enzyme linked immunological kit and its application of diclazuril Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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Abstract
The present invention provides a kind of enzyme linked immunological kits for detecting diclazuril, it includes: the ELISA Plate for being coated with diclazuril coupled antigen, diclazuril monoclonal antibody, enzyme label antiantibody, diclazuril standard solution, substrate developing solution, terminate liquid, cleaning solution, redissolves liquid.The invention also discloses a kind of methods using above-mentioned enzyme linked immunological kit detection diclazuril, it includes: to carry out sample pre-treatments first, is then detected with kit, ultimate analysis testing result.Enzyme linked immunological kit provided by the invention can be used for detecting the content of diclazuril in animal tissue, and easy to operate, 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 for detecting the ELISA reagent of diclazuril
Box, the detection particularly suitable for diclazuril content in animal tissue.
Background technique
Diclazuril is the non-polyether class chemical synthesis developed by Belgian Janssen Pharmaceutica the 1980s
Anticoccidial drug belongs to triazine benzene acetonitrile compound, has the characteristics that efficient, low toxicity, wide spectrum, is commonly used for aviculture and rabbit keeping
The prevention and treatment of coccidiosis.Diclazuril has inhibition or killing effect to aquatic animal sporozoite etc., in aquaculture
It is mainly used for preventing and treating the sporidiosis such as cyprinid fish Myxosporean, myxobolus, tail spore, quadrupole worm, monopole worm.Due to ground gram
Pearl benefit has the characteristics that low toxicity, wide spectrum, dosage are small, there is drug abuse in use, does not abide by situations such as off-drug period,
Therefore a series of residue of veterinary drug problems are brought, there are potentially hazardous to human health.
The residual marker of diclazuril is itself.National governments and international organization have formulated in animal derived food
The maximum residue limit (maximum residue limits, MRLs) of diclazuril.The Ministry of Agriculture of China announces No. 235 rule
Determining MRLs of the diclazuril in sheep/fowl/rabbit muscle, fat, liver and nephridial tissue is respectively 500,1000,3000,2000
μg/kg。
Currently, the detection method of diclazuril residual quantity mainly has gas chromatography-mass spectrography, high performance liquid chromatography, liquid
Phase chromatography-tandem mass spectrometry and high resolution mass spectrometry.These methods can simultaneously similar drugs such as gram pearl benefit it is how residual
It stays qualitative and quantitative analysis, but needs expensive instrument and special technical staff, sample pretreatment process is complex and expensive,
Time-consuming length, it is difficult to meet the needs that a large amount of samples and field sample quickly detect.Enzyme linked immunosorbent assay analysis method (ELISA) tool
Have the characteristics that it is easy quickly, it is special it is sensitive, sample capacity is big, analysis cost is low, can simplify or even save sample purification step
Suddenly, unique advantage is shown in great amount of samples and the quick selective mechanisms of field samples, can preferably meet China's food enterprise
The developments such as industry, government function supervision department detect work, with development potential.
Summary of the invention
It is simple, easy to use, cheap, portable for ground gram that the purpose of the present invention is to provide a kind of structures
The enzyme linked immunological kit of pearl benefit detection, and provide it is a kind of efficiently, it is accurate, easy, suitable for high-volume screening sample it is qualitative,
Quantitative detecting method.
Kit of the present invention, it includes: that the ELISA Plate for being coated with diclazuril coupled antigen, diclazuril monoclonal are anti-
Body, diclazuril standard solution, substrate developing solution, terminate liquid, cleaning solution, redissolves liquid at enzyme label antiantibody；The ground gram
Pearl benefit monoclonal antibody is prepared using diclazuril coupled antigen as immunogene, and the diclazuril coupled antigen is
It is obtained by diclazuril haptens with carrier protein couplet, the carrier protein is mouse haemocyanin, thyroprotein, ox blood
Pure albumen, rabbit serum proteins, human serum albumins, ovalbumin, hemocyanin or fibrinogen, the diclazuril
Haptens is to react to obtain with glacial acetic acid, hydrobromic acid by diclazuril, molecular structural formula are as follows:
The antiantibody of the enzyme label antiantibody is sheep anti mouse antiantibody.
The marker enzyme of the enzyme label antiantibody is horseradish peroxidase；The enzyme label antiantibody is using glutaraldehyde
What marker enzyme and antiantibody were coupled by method or Over-voltage protection.
In order to be more convenient on-site supervision and great amount of samples screening, the kit further include diclazuril standard solution,
Substrate developing solution, cleaning solution, redissolves liquid at terminate liquid.
5 bottles of the diclazuril standard solution, concentration are respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L.
The substrate developing solution is made of substrate solution A liquid and substrate solution B liquid, and the substrate solution A liquid is hydrogen peroxide or mistake
Urea is aoxidized, the substrate solution B liquid is o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is that the sulfuric acid of 1~2mol/L is molten
Liquid.
The cleaning solution is preferably that pH value is 7.4, contains 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ nitrine
Change the phosphate buffer of sodium, 0.1~0.3mol/L.
The liquid that redissolves is preferably the phosphate buffer that pH value is 7.0,0.1mol/L.
Wherein coating buffer used in ELISA Plate preparation process be pH value be 9.6, the carbonate of 0.05mol/L
Buffer, confining liquid are that pH value is 7.1~7.5, the phosphate-buffered containing 1%~3% casein, 0.1~0.3mol/L
Liquid.
The preparation process of ELISA Plate in the present invention are as follows: coating antigen is diluted to 20 μ g/mL with coating buffer, every hole is added
100 μ L, 37 DEG C are protected from light 2h or 4 DEG C of incubation overnight, and liquid in hole of inclining is washed 2 times with cleaning solution, and each 30s is patted dry, then
150~200 μ L confining liquids are added in every hole, 37 DEG C are protected from light 1~2h of incubation, and liquid pats dry in hole of inclining, and use aluminium film after dry
Vacuum sealing saves.
Testing principle of the invention are as follows:
The pre-coated diclazuril coupled antigen on capillary strip adds ground after sample solution or standard solution is added
Gram pearl benefit monoclonal antibody solution, coated diclazuril coupled antigen competition ground on the diclazuril and ELISA Plate in sample
Gram pearl benefit monoclonal antibody is added enzyme label antiantibody and amplifies effect, developed the color with developing solution, sample absorbance value with
The content of gram pearl benefit is negatively correlated, and the residual quantity of diclazuril in sample can be obtained compared with standard curve；Simultaneously according to enzyme
The depth of color on target, compared with the standard solution color of series of concentrations can in rough judgement sample diclazuril it is residual
The concentration range of allowance.
The present invention also provides a kind of methods using above-mentioned enzyme linked immunological kit detection diclazuril residual quantity, it is wrapped
Include step:
(1) Sample pretreatment；
(2) it is detected with kit；
(3) analysis detection result.
The enzyme linked immunological kit that the present invention detects diclazuril mainly uses indirect competitive ELISA method qualitative or quantitative
Detect the residual quantity of diclazuril in sample；Low to the pre-treatment requirement of sample, Sample pretreatment process is simple, can be fast simultaneously
Speed detection high-volume sample；Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, have specificity high, clever
The features such as sensitivity is high, accuracy is high, accuracy is high.Enzyme linked immunological kit of the invention, structure is simple, easy to use, price
Cheaply, carrying convenience, detection method efficiently, it is accurate, easy, suitable for the qualitative and quantitative analysis of high-volume screening sample.
Detailed description of the invention
Fig. 1: diclazuril hapten synthesis route map
Fig. 2: kit standard curve graph
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 be not intended to limit the scope of the invention.
The preparation of 1 reagent constituents of embodiment
1, the synthesis of diclazuril haptens (synthetic route is shown in attached drawing 1)
Diclazuril 1g is taken, ice acetic acid 5mL dissolution clarification adds hydrobromic acid aqueous solution 20mL, and 100 DEG C of heating, stirring is instead
Answer 2h.Stop reaction, add water 100mL, 1mol/L NaOH is added to be neutralized to pH 6, add ethyl acetate 200mL × 3, extracts three times,
Merging organic phase, 20g anhydrous sodium sulfate is dry, it is evaporated, upper silicagel column, ethyl acetate/petroleum ether (v/v, 1/1) elution separation,
Obtain carboxyl diclazuril haptens product 0.91g, yield 87.5%.
2, the synthesis and identification of diclazuril coupled antigen
Immunogene preparation --- diclazuril haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
Carboxyl diclazuril haptens 16mg is taken, n,N-Dimethylformamide 0.5mL is added to dissolve, adds N- hydroxysuccinimidyl acyl sub-
Amine (NHS) 6mg, carbodiimides (EDC) 12mg stir 2h, obtain haptens activating solution A liquid；BSA 50mg is taken, is added
0.1mol/L sodium bicarbonate solution 3mL, dissolution clarification, obtains B liquid；A liquid is slowly dropped in B liquid, 4 DEG C of stirring 4h,
0.02mol/L PB buffer dialysis purification 3d, is changed liquid 3 times daily, and packing obtains diclazuril haptens-BSA immunogene ,-
20 DEG C save backup.
Coating antigen preparation --- diclazuril haptens and ovalbumin (OVA) coupling obtain coating antigen.
Carboxyl diclazuril haptens 9mg is taken, adds n,N-Dimethylformamide 0.4mL to dissolve, adds NHS 5mg, EDC
9mg stirs 2h, obtains haptens activating solution A liquid；OVA 50mg is taken, 0.1mol/L sodium bicarbonate solution 3mL is added, is dissolved clear
Clearly, B liquid is obtained；A liquid is slowly dropped in B liquid, 4 DEG C of stirring 4h, 0.02mol/L PB buffer dialysis purification 3d, daily
It changes liquid 3 times, dispenses, obtain diclazuril haptens-OVA coating antigen, -20 DEG C save backup.
In the ratio of synthesis diclazuril coupled antigen reaction haptens used, carrier protein and coupled product, carry out purple
(200nm~400nm) sweep measuring outside, by comparing three respectively the light absorption value of 260nm and 280nm calculate its combine than.
The absorption maximum of the maximum absorption band of conjugate diclazuril hapten-carrier albumen and diclazuril haptens, carrier protein
Peak shows that the synthesis of diclazuril hapten-carrier albumen is successful compared to apparent variation has occurred.
3, the preparation of diclazuril monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: diclazuril haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is abundant
The Balb/c mouse of 6 week old, every 0.2mL is subcutaneously injected in emulsification；
2) booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, is replaced with not formula Freund's incomplete adjuvant
Freund's complete adjuvant, method and the same first immunisation of dosage；
3) potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reaches 1:
Following final immunization is carried out when 10000 or more: the immunogen solution 0.1mL of any adjuvant is not added in intraperitoneal injection, puts to death after three days
Mouse takes its spleen to merge with myeloma cell；
4) cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Utilize limiting dilution
Method carries out cloning to positive hole, obtains and establishes the hybridoma cell strain of stably excreting diclazuril monoclonal antibody, take place
Cell suspension is made with frozen stock solution in the hybridoma of logarithmic growth phase, is sub-packed in cryopreservation tube, is saved for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery: diclazuril monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, 37 DEG C of water-baths are immediately placed in
Middling speed is melted, and after centrifugation removal frozen stock solution, moves into culture culture in glassware；
2) ascites and antibody purification are prepared: using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing stone
Only, hybridoma 5 × 10 is injected intraperitoneally in wax oil 0.5mL/ after 7 days5A/only, ascites is acquired after 7 days.With octanoic acid-saturation sulphur
Sour ammonium method is purified, and diclazuril monoclonal antibody solution (- 20 DEG C of preservations) is obtained.
(3) measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(100000~300000).
Indirect competitive ELISA method: using diclazuril haptens-OVA conjugate coated elisa plate, and diclazuril mark is added
The sheep anti mouse antiantibody solution of quasi- product solution, diclazuril monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C anti-
30min is answered, liquid in hole is poured out, is washed 3~5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of reactions are added
After 15min, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at wavelength 450nm.
(4) measurement of monoclonal antibody specificity
Antibody specificity refers to the ability of its homospecificity antigen binding and the ratio with such antigen-analogues ability
Compared with common cross reacting rate is as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
Diclazuril, toltrazuril, coban, Madumycin, salinomycin are serially diluted by this experiment, respectively with list
Clonal antibody carries out indirect competitive ELISA, makes standard curve, and analysis obtains IC50, cross reacting rate is then calculated as follows:
The cross reacting rate of each analog as the result is shown are as follows: diclazuril 100%, toltrazuril 11.4%, coban
< 0.1%, Madumycin < 0.1%, salinomycin < 0.1%.Antibody of the present invention is mould to toltrazuril, coban, horse Du
The analogs no cross reaction such as element, salinomycin, has specific binding just for diclazuril.
4, the preparation of sheep anti mouse antiantibody
It is that immune animal obtains sheep anti mouse antiantibody using source of mouse antibody as immunogen immune pathogen-free domestic sheep with sheep.
5, the preparation of enzyme label antiantibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.It passes
It is 4:1 that the Over-voltage protection of system, which requires the molar concentration rate of enzyme and antibody in reaction system, since horseradish peroxidase is strong
Many sites in conjunction with antibody are generated under oxidation, it is each that the horseradish peroxidase molecule activated in this way acts as connection
The bridge of molecule reduces the enzymatic activity of enzyme marker, makes to be mixed with many condensates in the conjugate of preparation.In order to solve this
A problem, we are improved traditional method, it may be assumed that
(1) closed process of amino is eliminated, because the amino that can generate the connection of itself amino is actually seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, and the method after improvement is than traditional side
Method is easy, reduces to the loss of enzymatic activity.
6, the preparation of ELISA Plate
Coating antigen (diclazuril haptens-OVA conjugate) is diluted to 20 μ g/mL with coating buffer, every hole is added
100 μ L, 37 DEG C are protected from light incubation 2h, and liquid in hole of inclining is washed 2 times, each 30s is patted dry, then in every hole with cleaning solution
200 μ L confining liquids are added, 37 DEG C are protected from light incubation 2h, and liquid pats dry in hole of inclining, and are saved after dry with aluminium film vacuum sealing.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of diclazuril
The enzyme linked immunological kit for setting up detection diclazuril, makes that it includes following components:
(1) it is coated with the ELISA Plate of diclazuril coupled antigen；
(2) 5 bottles of diclazuril standard solution, concentration are respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L；
(3) diclazuril monoclonal antibody working solution；
(4) the sheep anti mouse antiantibody of horseradish peroxidase-labeled is used；
(5) substrate developing solution is made of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) it is 7.4 that cleaning solution, which is pH value, containing 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide,
The phosphate buffer of 0.1~0.3mol/L；
(8) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.1mol/L.
The detection of diclazuril in 3 sample of embodiment
1, Sample pretreatment
1.0g ± 0.05g tissue samples are weighed into 50mL centrifuge tube, 1mL 0.1mol/L sodium hydroxide is added, uses whirling motion
Instrument whirling motion 1min, adds 7mL acetonitrile, with (20~25 DEG C) centrifugation 5min of whirling motion instrument whirling motion 2min, 3000g room temperature；It pipettes
1mL upper organic phase dries up under 50~60 DEG C of water-bath nitrogen streams into 10mL clean dried glass tube；1mL is added to redissolve
Liquid, with vortex instrument whirling motion 30s；Take 50 μ L for analyzing.
2, it is detected with kit
Diclazuril standard solution or premenstrual place are added into the micropore of enzyme marker plate for being coated with diclazuril coupled antigen
Then 50 hole μ L/ of sample solution of reason is added 50 hole μ L/ of sheep anti mouse antiantibody of horseradish peroxidase-labeled, adds ground
Gram 50 hole μ L/ of pearl benefit monoclonal antibody working solution, gently oscillation mixes, with anti-in cover board membrane cover plate 25 DEG C of light protected environments of postposition
Answer 30min；Liquid in hole is poured out, every hole is added 250 μ L cleaning solutions and sufficiently washs 4~5 times, and every minor tick 10s uses blotting paper
It pats dry；50 μ L of substrate solution A liquid urea peroxide, substrate solution B liquid tetramethyl benzidine (TMB) 50 μ L is added in every hole, and gently oscillation is mixed
Even, with 15min is reacted in cover board membrane cover plate 25 DEG C of light protected environments of postposition, 50 μ L of terminate liquid 2mol/L sulfuric acid is added in every hole, 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
Standard) absorbance value (B0) multiplied by 100%, obtain percentage absorbance value.With diclazuril standard concentration (μ g/L)
Logarithm is X-axis, and percentage absorbance value is Y-axis, draws standard curve, as shown in Figure 2.It is molten that sample is calculated with same method
The diclazuril content of the percentage absorbance value of liquid, each corresponding sample can then be read from standard curve.
4 diclazuril enzyme linked immunological kit technical parameter of embodiment determines test
1, kit sensitivity and detection limit
Conventionally assay kit sensitivity, kit standard curve minimum point are 1 μ g/L, the model of standard curve
It encloses for 1~27 μ g/L, IC50(50% inhibition concentration) floating range is 2.5~4.5 μ g/L；It is each to blank pork, chicken sample
20 parts are detected, and the concentration corresponding to each percentage absorbance value is found from standard curve, with being averaged for 20 parts of concentration of specimens
Being worth indicates detection limit plus 3 times of standard deviations, as a result obtains this method and is limited to 8 μ g/kg to the detection of tissue samples.
2, sample preci-sion and accuracy is tested
It is inclined with the testing result relative standard of a certain concentration samples of replication using the rate of recovery as accuracy estimating index
Poor (RSD%) is used as precision evaluation index.Calculation formula are as follows: the rate of recovery (%)=actual measured value/theoretical value × 100%,
Wherein theoretical value is the addition concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, X
For the average value of determination data.
Blank pork, chicken sample are added back by 8 μ g/kg, 16 μ g/kg, 32 μ g/kg, tri- concentration diclazurils
Measurement is received, each sample does 4 and is measured in parallel with three batches of different kits, calculates the sample mean rate of recovery and precision
As a result it see the table below.
1 precision of table and accuracy test
Blank pork, chicken sample are added with the diclazuril of 8,16,32 tri- concentration of μ g/kg, it is average to recycle
Rate is between 80%~110%；Relative standard deviation is respectively less than 15% in batch, between criticizing.
3, stabilization of kit is tested
Kit preservation condition is 2~8 DEG C, by measurement in 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, diclazuril addition actual measured value are within normal range (NR).Consider in transport and use process,
It has improper preservation condition to occur, kit is placed 7 days under 37 DEG C of preservation conditions, carry out accelerated aging tests, as a result
Show that the kit indices comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C
Refrigerator freezing 7 days, measurement result also indicated that kit indices are completely normal.It can show that kit can be with from result above
At least saved 12 months at 2~8 DEG C or more.
Claims (6)
1. it is a kind of detect diclazuril enzyme linked immunological kit, comprising: be coated with diclazuril coupled antigen ELISA Plate,
It is gram pearl benefit monoclonal antibody, enzyme label antiantibody, diclazuril standard solution, substrate developing solution, terminate liquid, cleaning solution, multiple
Solution；It is characterized in that the diclazuril monoclonal antibody is prepared using diclazuril coupled antigen as immunogene,
The diclazuril coupled antigen is to be obtained by diclazuril haptens with carrier protein couplet, and the carrier protein is mouse serum
Albumen, thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human serum albumins, ovalbumin, hemocyanin or fiber
Proteinogen, the diclazuril haptens are to react to obtain with glacial acetic acid, hydrobromic acid by diclazuril, molecular structural formula are as follows:
2. kit as described in claim 1, it is characterised in that the antiantibody of the enzyme label antiantibody is that sheep anti mouse is anti-
Body.
3. kit as described in claim 1, it is characterised in that the marker enzyme of the enzyme label antiantibody is horseradish peroxidating
Object enzyme, the substrate developing solution are made of substrate solution A liquid and substrate solution B liquid, and the substrate solution A liquid is hydrogen peroxide or peroxidating
Urea, the substrate solution B liquid are o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is the sulfuric acid solution of 1~2mol/L.
4. kit as described in claim 1, it is characterised in that the cleaning solution is that pH value is 7.4, containing 0.5%~
1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide, 0.1~0.3mol/L phosphate buffer；The redissolution liquid is
PH value is the phosphate buffer of 7.0,0.1mol/L.
5. kit as described in claim 1, it is characterised in that the concentration of the diclazuril standard solution is respectively 0 μ
g/L、1μg/L、3μg/L、9μg/L、27μg/L。
6. a kind of method of diclazuril residual quantity in test sample, comprising steps of
(1) sample pre-treatments；
(2) it is detected with the described in any item kits of Claims 1 to 5；
(3) analysis detection result.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110058008A (en) * | 2019-04-15 | 2019-07-26 | 广东省疾病预防控制中心 | The detection method and its application of bongkrekic aicd in a kind of food |
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