CN105044325B - The enzyme linked immunological kit of detection triazophos and application thereof - Google Patents
The enzyme linked immunological kit of detection triazophos and application thereof Download PDFInfo
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Abstract
The invention provides a kind of enzyme linked immunological kit detecting triazophos, it includes: be coated with the ELISA Plate of coating antigen, triazophos standard solution, ELIAS secondary antibody, triazophos specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, described coating antigen is triazophos coupled antigen, and described ELIAS secondary antibody is the sheep anti mouse anti antibody of enzyme labelling.The invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit detection triazophos, it includes: first carries out sample pre-treatments, then detects with test kit, ultimate analysis testing result.The enzyme linked immunological kit that the present invention provides can be used for detecting the content of triazophos in vegetable sample, it is easy and simple to handle, low cost, highly sensitive, can on-site supervision and the examination of applicable great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detecting triazophos, can determine
The residual quantity of triazophos medicine in property, detection by quantitative vegetable.
Background technology
Triazophos, has another name called triazophos, and English common name Triazophos, chemical name O, O-diethyl-O-(1-phenyl-1,2,4-
Triazole-3-base) thiophosphate.Sterling is sundown liquid, and when 20 DEG C, the dissolubility in water is 30～40mg/L, dissolves in
Most of organic solvents；Stable to light, hydrolyze in acid, alkaline media, 140 DEG C of decomposition.Triazophos has strong stomach function regulating of tagging
Toxic action, good disinsection effect, ovicidal action is obvious, and permeability is relatively strong, without systemic action, is that a kind of medium poison, wide spectrum are organic
Phosphorus pesticide, acaricide, have certain nematocidal effect concurrently, step on the crops such as China Oryza sativa L., Cotton Gossypii, vegetable and Fructus Mali pumilae
Note, is mainly used in preventing and treating the many important pests on the staple crops such as grain, Cotton Gossypii, fruit tree, vegetable.Due to triazophos
Applying effective, its toxicity of high-toxic pesticide such as relative Bayer 71628 is low, therefore, applies wide on crops.
But, a large amount of use of triazophos also brings residue problem to some edible agricultural product and environment.Triazophos belongs to organophosphor
Pesticide, moderate toxicity, the multi viscera such as nerve, liver, kidney, the heart, lung and reproductive system all there are is obvious toxic and side effects, wherein
Poison dominant mechanism is the activity of suppression acetylcholine esterase.European Union's regulation triazophos MRL (maximum residue limit,
MRL) must not be the pesticide species detected；Regulation in Japan's " Positive List System ", except Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), jade
Rice, Semen Fagopyri Esculenti, other Cereals, Semen Gossypii and Oleum Gossypii semen set MRL, and remaining is suitable for " uniform limit ", i.e. 0.01mg/kg；
China's national standard " GB 2763-2005 Pesticide MRL " limits the MRL of triazophos as Oryza glutinosa
0.05mg/kg, Semen Gossypii 0.1mg/kg, do not formulate the MRL standard of triazophos in vegetable.Therefore, triazophos is set up
The Fast Detection Technique of residual, strengthens monitoring, scientifical use triazophos, for ensureing human health and food safety, reduces ring
Environment pollution, reduces significant on the impact that agricultural byproducts outlet produces etc..
The method of triazophos in vegetable that measures provided in China's standards system is all multi-residue determination method, has national standard
(GB/T 19648-2006、GB/T 5009.218-2008、GB/T 20769-2008、GB/T 23204-2008、GB/T
23205-2008, GB/T 23376-2009), industry standard (NY/T 1379-2007, NY/T 761-2008, SN/T 1950-2007,
SN/T 0148-2011) and provincial standard (DB34/T 1076-2009), the detection method related to has liquid chromatography, gas phase
Chromatography and chromatograph-mass spectrometer coupling method.Instrumental method has the advantages such as highly sensitive, result is accurate, but fund and personnel etc. throw
Enter relatively costly.Present invention application euzymelinked immunosorbent assay (ELISA), measures the residual quantity of triazophos medicine in vegetable, has detection limit low, special
The opposite sex is strong, easy and simple to handle, speed is fast, testing cost is low in detection, is very easy to the advantages such as popularization.
Summary of the invention
It is an object of the invention to provide and a kind of can detect the enzyme linked immunological kit of triazophos drug residue in vegetable, and carry
For a kind of efficiently, accurately, easy, be suitable to the qualitative and quantitative analysis method of batch samples screening.
Test kit of the present invention, it includes: be coated with the ELISA Plate of coating antigen, triazophos standard solution, ELIAS secondary antibody, triazole
Phosphorus specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, described coating antigen is triazophos coupled antigen, institute
State the sheep anti mouse anti antibody that ELIAS secondary antibody is enzyme labelling.
Described triazophos coupled antigen is to be obtained with carrier protein couplet by triazophos hapten, and described triazophos hapten is by three
Azoles phosphorus and excess acetyl chloride obtain, and described carrier protein can be Mus serum albumin, thyroprotein, bovine serum albumin, Sanguis Leporis seu oryctolagi
Albumin, human albumin, ovalbumin, hemocyanin or Fibrinogen.
Described triazophos specific antibody is to prepare using triazophos coupled antigen as immunogen, and described triazophos specificity resists
Body can be triazophos monoclonal antibody or triazophos polyclonal antibody, wherein preferred triazophos monoclonal antibody.
The marker enzyme of described enzyme marker is horseradish peroxidase or antibacterial extraction alkaline phosphatase, wherein preferred Radix Cochleariae officinalis peroxide
Compound enzyme；ELIAS secondary antibody is to use the Over-voltage protection after improveing to carry out coupling to obtain.
For more convenient on-site supervision and great amount of samples examination, described test kit also includes that triazophos standard solution, substrate develop the color
Liquid, stop buffer, cleaning mixture, redissolution liquid.
Described triazophos standard solution 6 bottles, concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L.
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and A is
Hydrogen peroxide or urea peroxide, B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is sulphuric acid or the liquid of 1～2mol/L
Hydrochloride buffer；When marker enzyme is antibacterial extraction alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer,
Described stop buffer is 1～2mol/L sodium hydroxide solution.
Described cleaning mixture preferably pH value is 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives,
The phosphate buffer of 0.1～0.3mol/L, described percentage ratio is percent weight in volume.
Described redissolution liquid preferably pH value is 7.0, the phosphate buffer of 0.02mol/L, and described percentage ratio is weight volume basis
Ratio.
Wherein in ELISA Plate preparation process used be coated buffer be pH value be 9.6, the carbonate buffer solution of 0.05mol/L,
Confining liquid be pH value be 7.1～7.5, containing 1%～3% casein, 0.1～the phosphate buffer of 0.3mol/L, described percentage ratio
For percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: with being coated buffer, coating antigen being diluted to 20 μ g/mL, every hole adds 100 μ l,
25 DEG C of lucifuges hatch 2h or 4 DEG C overnight, and liquid in hole of inclining washs 2 times with cleaning mixture, each 30s, pats dry, then exist
Adding 150～200 μ l confining liquids in every hole, 25 DEG C of lucifuges hatch 1～2h, and in hole of inclining, liquid pats dry, dried by aluminum film vacuum
Seal and preserve.
The Cleaning Principle of the present invention is:
This test kit uses indirect competitive ELISA method, pre-coated coupled antigen on ELISA Plate capillary strip, residual in sample
The antibody of the coupled antigen anti-triazophos of competition that triazophos is pre-coated with on capillary strip, after adding ELIAS secondary antibody, uses tmb substrate
Colour developing, sample light absorption value with its contained by the content of triazophos become negative correlation, be multiplied by its corresponding dilution more again with standard curve again
Number, can draw the residual quantity of triazophos in sample.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection triazophos, it includes step:
(1) sample pre-treatments；
(2) detect with test kit；
(3) testing result is analyzed.
The present invention detects the enzyme linked immunological kit of triazophos and mainly uses ELISA method qualitatively or quantitatively to detect triazophos in sample
Content；Pre-treatment to sample requires low, and sample pretreatment process is simple, can quickly detect batch samples simultaneously；Mainly
Reagent provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accurate
Degree high.The enzyme linked immunological kit of the present invention, simple in construction, easy to use, low price, carrying convenience, detection
Method is efficient, accurate, easy, be suitable to the qualitative, quantitative of batch samples screening.
Accompanying drawing explanation
Fig. 1: triazophos hapten synthesis route map
Fig. 2: triazophos hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3: kit standard curve chart
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention,
And it is not limited to the scope of the present invention.
The preparation of embodiment 1 reagent constituents
1, the haptenic preparation of triazophos
Adding 10mL dichloromethane and 0.5g triazophos in 100mL there-necked flask, be cooled to-5 DEG C, the lower addition of stirring 1.01 is worked as
The chloroacetic chloride of amount, temperature control adds the aluminum chloride of 2 equivalents, after temperature control 5 DEG C reacts 6 hours, adds dilute hydrochloric acid and frozen water, acetic acid
Ethyl ester extracts, and washing, anhydrous magnesium sulfate is dried organic facies, evaporated under reduced pressure solvent, and petroleum ether-ethyl acetate system recrystallization obtains second
Acylate.
Walk acetylate, 10mL pyridinium dissolution on 100mL there-necked flask adds, add the carboxymethyl azanol of 1.2 equivalents, 65 DEG C
Reacting 5 hours, ethyl acetate extracts, washing, and anhydrous magnesium sulfate is dried organic facies, decompression distillation, obtains hapten, and two steps are received
Rate 58%.
Take above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as in figure 2 it is shown, the carboxyl signal peak of 11.0ppm, 2.85ppm methyl letter
The appearance at number peak, illustrates hapten synthesis success.
2, the preparation of antigen
Immunogen is prepared triazophos hapten and is obtained immunogen with bovine serum albumin (BSA) coupling.
Take 8.5mg hapten, be dissolved in 1mL DMF, take 30mg EDC and after NHS 0.2mL water fully dissolves,
Add in (1), stir 24h under room temperature, i.e. can get reactant liquor (1).Weigh BSA50mg, be allowed to be substantially dissolved in 3.8mL
In PB (PH 9.0), reactant liquor (1) is dropwise slowly dropped in protein solution, and stirs 24h at room temperature.Use 0.01mol/L
PBS 4 DEG C dialysis 3d changes 3 dialysis solution, to remove unreacted small-molecule substance every day.Subpackage, saves backup in-20 DEG C.
Coating antigen is prepared triazophos hapten and is obtained immunogen with ovalbumin (OVA) coupling.
Take 8.5mg hapten, be dissolved in 1mL DMF, take 30mg EDC and after NHS 0.2mL water fully dissolves,
Add in (1), stir 24h under room temperature, i.e. can get reactant liquor (1).Weigh OVA 50mg, be allowed to be substantially dissolved in 3.8mL
In PB (PH 9.0), reactant liquor (1) is dropwise slowly dropped in protein solution, and stirs 24h at room temperature.Use 0.01mol/L
PBS 4 DEG C dialysis 3d changes 3 dialysis solution, to remove unreacted small-molecule substance every day.Subpackage, saves backup in-20 DEG C.
3, the preparation of triazophos monoclonal antibody
Animal immune: immunogen above-mentioned steps obtained is injected in Balb/c Mice Body, immunizing dose is 150 μ g/, makes
It produces antiserum.
Cell merge and cloning: after mice serum measurement result is higher, take its splenocyte, in 8:1 (quantitative proportion) ratio with
SP2/0 myeloma cell fusion, uses indirect competitive ELISA to measure cell supernatant, the positive hole of screening.Utilize limiting dilution
Method carries out cloning to positive hole, until obtaining secreting the hybridoma cell strain of triazophos monoclonal antibody.
Cell cryopreservation and recovery: monoclonal hybridoma strain frozen stock solution is made 1 × 106The cell suspension of individual/mL, at liquid
The medium-term and long-term preservation of nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into training
Support culture in glassware.
The production of monoclonal antibody and purification: Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5mL/ only, 7 days pneumoretroperitoneum notes
Penetrate stable monoclonal hybridoma strain 5 × 105Individual/only, gather ascites after 7 days.Ascites is carried out by octanoic acid-saturated ammonium sulfate method
Purification ,-20 DEG C of preservations.
4, the preparation of enzyme mark disome
Over-voltage protection after sheep anti mouse anti antibody is used improvement with horseradish peroxidase (HRP) carries out coupling.
5, the preparation of ELISA Plate
With being coated buffer, coating antigen being diluted to 20 μ g/mL, every hole adds 100 μ l, and 25 DEG C of lucifuges are hatched 2h, inclined in hole
Liquid, washs 2 times with cleaning mixture, each 30s, pats dry, then add 200 μ l confining liquids in every hole, and 25 DEG C of lucifuges are hatched
2h, in hole of inclining, liquid pats dry, and dried sealing by aluminum film vacuum preserves.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of triazophos
Set up the enzyme linked immunological kit of detection triazophos so that it is comprise following component:
(1) ELISA Plate of triazophos coupled antigen it is coated；
(2) triazophos standard solution 6 bottles, concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L；
(3) with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(4) triazophos specific antibody；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) stop buffer is 2mol/L sulphuric acid；
(7) cleaning mixture be pH value be 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives,
The phosphate buffer of 0.1～0.3mol/L, described percentage ratio is percent weight in volume；
(8) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.02mol/L, described percentage ratio is percent weight in volume.
The detection of triazophos in embodiment 3 vegetable
1, sample pre-treatments
With homogenizer homogeneous structure sample；Weigh the equal pledge of 2.0 ± 0.05g in 10ml polystyrene centrifuge tube, be separately added into 2ml
0.1M sodium hydroxide solution, 10ml ethyl acetate, vibrate 5min with agitator, be sufficiently mixed；More than 3000g, room temperature (20-25 DEG C
/ 68-77) centrifugal 5min；Pipette 1ml upper organic phase in 10ml clean dried teat glass；In 50-60 DEG C of (122-140
) water-bath flows down nitrogen and dry up；Add 1ml redissolution working solution, whirling motion 1min.Take 200 μ l and join 1800 μ l redissolution work
In liquid, it is sufficiently mixed；Take 50 μ l for analyzing.
2, detect with test kit
Adding standard substance/sample 50 μ l in corresponding micropore, add antibody working solution 50 μ l/ hole, mixing of vibrating gently, with lid
The rearmounted 25 DEG C of light protected environment of plate membrane cover plate react 30min.Carefully open cover plate film, liquid in hole is dried, with washing work
Liquid 250 μ l/ hole, fully washing 4-5 time, every minor tick 10s, pat dry with absorbent paper.Add ELIAS secondary antibody 100 μ l/ hole, gently
Vibration mixing, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.Carefully open cover plate film, liquid in hole is got rid of
Dry, with wash operating solution 250 μ l/ hole, fully washing 4-5 time, every minor tick 10s, pat dry with absorbent paper.Add substrate solution A
Liquid 50 μ l/ hole, adds substrate solution B liquid 50 μ l/ hole, mixing of vibrating gently, keeps away with cover plate membrane cover plate rearmounted 25 DEG C (77)
Luminous environment reacts 15min.Adding stop buffer 50 μ l/ hole, mixing of vibrating gently, setting microplate reader, at 450nm, measures every
Hole OD value.
3, Analysis of test results
The percentage absorptance of standard substance or sample is equal to the meansigma methods (diplopore) of the absorbance of standard substance or sample divided by first
The meansigma methods of the absorbance of standard substance (0 standard), then it is multiplied by 100%, obtain the percentage absorbance of standard substance or sample.
With standard substance percentage absorptance as vertical coordinate, with the logarithm of triazophos standard concentration (μ g/L) as abscissa, draw standard bent
Line chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by it
Corresponding extension rate is the actual concentrations of triazophos in sample.
The determination test of embodiment 4 triazophos technical parameter
1, test kit sensitivity and detection limit
Conventionally measuring test kit sensitivity, standard curve is in the range of 1～81 μ g/L, IC50(50% inhibition concentration)
Domain of walker is 4～6 μ g/L；Detecting 20 parts of samples, that finds corresponding to each percentage absorbance from standard curve is dense
Degree, represents detection limit with the meansigma methods of 20 parts of concentration of specimens plus 3 times of standard deviations, and result shows, the method detection to vegetable
Limit is 50 μ g/kg.
2, sample preci-sion and accuracy test
Using the response rate as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) as precision evaluation index.Computing formula is: the response rate (%)=practical measurement value/theoretical value × 100%,
The wherein interpolation concentration of theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation,
X is the meansigma methods of determination data.
It is added cabbages leaves respectively reclaiming measuring by the triazophos of 100 μ g/kg, 200 two concentration of μ g/kg, each sample
Do 4 parallel, be measured with three batches of different reagent, calculate the average recovery rate of sample and precision result see table.
Table 1 Chinese cabbage sample precision and accuracy test
Being added Chinese cabbage respectively with the triazophos of 100 μ g/kg, 200 two concentration of μ g/kg, average recovery rate exists
Between 75.4%～81.9%；Batch in, batch between relative standard deviation be respectively less than 10%.
3, stabilization of kit test
Test kit preservation condition is 2～8 DEG C, through the mensuration of 12 months, and the maximum absorbance value (zero standard) of test kit, 50%
Inhibition concentration, triazophos add practical measurement value all within normal range.Consider, during transport and using, to have anon-normal
Often preservation condition occurs, is placed 7 days by test kit, be accelerated senile experiment under 37 DEG C of preservation conditions, and result shows this examination
Agent box indices complies fully with requirement.Occur in view of test kit freezing situation, test kit put into-20 DEG C of refrigerator freezings 7 days,
Measurement result also indicates that test kit indices is the most normal.Can show that test kit at least can preserve at 2～8 DEG C from result above
More than 12 months.
Claims (5)
1. the enzyme linked immunological kit detecting triazophos, it is characterised in that including: be coated with the ELISA Plate of coating antigen, triazole
Phosphorus standard solution, ELIAS secondary antibody, triazophos specific antibody, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid, institute
Stating coating antigen is triazophos coupled antigen, and described ELIAS secondary antibody is the sheep anti mouse anti antibody of enzyme labelling, it is characterised in that described triazole
Phosphorus coupled antigen is to be obtained with carrier protein couplet by triazophos hapten, and described carrier protein is Mus serum albumin, thyroid egg
In vain, bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or Fibrinogen, described three
The haptenic preparation method of azoles phosphorus is as follows: add 10mL dichloromethane and 0.5g triazophos, cooling in 100mL there-necked flask
To-5 DEG C, the lower chloroacetic chloride adding 1.01 equivalents of stirring, temperature control adds the aluminum chloride of 2 equivalents, and temperature control 5 DEG C reacts 6 hours
After, adding dilute hydrochloric acid and frozen water, ethyl acetate extracts, and washing, anhydrous magnesium sulfate is dried organic facies, evaporated under reduced pressure solvent, oil
Ether-ethyl acetate system recrystallization obtains acetylate, adds and walk acetylate in 100mL there-necked flask, and 10mL pyridine is molten
Solving, add the carboxymethyl azanol of 1.2 equivalents, 65 DEG C are reacted 5 hours, and ethyl acetate extracts, and washing, anhydrous magnesium sulfate has been dried
Machine phase, decompression distillation, obtain hapten, two step yields 58%.
2. test kit as claimed in claim 1, it is characterised in that the haptenic molecular structural formula of described triazophos is:
3. test kit as claimed in claim 1, it is characterised in that described triazophos specific antibody is with triazophos coupled antigen
Preparing as immunogen, described triazophos specific antibody is triazophos monoclonal antibody or triazophos polyclonal antibody.
4. test kit as claimed in claim 1, it is characterised in that the concentration of described triazophos standard solution be respectively 0 μ g/L,
1μg/L、3μg/L、9μg/L、27μg/L、81μg/L。
5. detect a method for triazophos content in sample, including step:
(1) sample pre-treatments；
(2) detect with the test kit described in any one of Claims 1 to 4；
(3) testing result is analyzed.
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| CN107807237A (en) * | 2017-12-13 | 2018-03-16 | 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 organophosphor |
| CN110133306B (en) * | 2019-05-09 | 2023-04-07 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting cimaterol and application thereof |
| CN112014569B (en) * | 2020-08-20 | 2023-11-10 | 金华科生物技术河北有限公司 | Myocardial type fatty acid binding protein determination kit |
| CN112898343A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院蔬菜花卉研究所 | Hapten of organophosphorus triazophos pesticide and preparation method thereof |
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| CN101477123B (en) * | 2009-01-13 | 2013-05-01 | 华南农业大学 | ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide |
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