CN109682961A - A kind of test strips and its application detecting cyphenothrin - Google Patents
A kind of test strips and its application detecting cyphenothrin Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
The invention discloses a kind of test strips and its application for detecting cyphenothrin.Test strips include sample absorption pad (1), conjugate release pad (2), reaction film (3), water absorption pad (4) and bottom plate (7), have on the reaction film and be coated with the detection line (5) of cyphenothrin hapten-carrier protein conjugate and be coated with the nature controlling line (6) of sheep anti mouse antiantibody, the conjugate release pad (2) is coated with cyphenothrin monoclonal antibody-colloid gold label object.The present invention also provides the remaining methods of cyphenothrin in a kind of crops such as the above-mentioned cyphenothrin test strips detection veterinary antibiotics of application.Test strips provided by the present invention have the characteristics that easy to operate, high sensitivity, detection speed are fast, at low cost, are suitble to the screening and on-site supervision of great amount of samples.
Description
Technical field
The present invention relates to a kind of test strips and its application for detecting cyphenothrin, and in particular to one kind is for detecting phenylate
The colloidal gold strip of Cyano chrysanthemate, it is especially suitable for the remaining detections of cyphenothrin in the crops such as veterinary antibiotics.
Background technique
Cyphenothrin, trade name Gokilaht match go out spirit, d cyphenothrin etc.；Molecular formula C24H25NO3, mainly
For preventing and treating domestic hygiene pest and storage pest.Its toxicity is very low, is that only allowance uses in civil aircraft in the U.S.
Insecticide, WHO approves and prevents and treats mosquito, the pests such as fly, 0.4% dextrorotation in cabin 2% d-phenothrin aerosol
Cyphenothrin pulvis can be directly used for human body prevention and treatment head louse.D-phenothrin can be processed to aerosol, oil emu,
Aqueous emulsion and pulvis, 10% aqueous emulsion can be used for groups' facility such as barracks, restaurant and train and the high place of security requirement.
Since different proportions plays the role of different, while hygienic insecticide is the sanitary insect pests such as prevention and treatment mosquito, fly and cockroach
Chemical products, be mainly used in the living environment of human living, be directly related to the health and life security of people.It is right
It is more in older generations' hygienic insecticide analysis of effective component document such as tetramethrin, cypermethrin, Permethrin, and p-phenylene cyanogen chrysanthemum
The a new generation such as ester, Imiprothrin hygienic insecticide analysis of effective component document is less, and mostly instrumental method.The foundation of this method
It is beneficial to improve regulatory measure and Supervision Level.
Summary of the invention
The object of the present invention is to provide a kind of high sensitivities, cyphenothrin easy to operate, at low cost, detection time is short
Residue detection test strips.
The remaining test strips of detection cyphenothrin provided by the present invention, including the release of sample absorption pad (1), conjugate
Pad (2), reaction film (3), water absorption pad (4) and bottom plate (7)；Have on the reaction film and is coated with cyphenothrin haptens-load
The detection line (5) of body protein conjugate and the nature controlling line (6) for being coated with sheep anti mouse antiantibody, conjugate release pad (2) spray
It is coated with cyphenothrin monoclonal antibody-colloid gold label object.
The cyphenothrin hapten-carrier protein conjugate is by cyphenothrin haptens and carrier protein couplet
It obtains, the carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
The cyphenothrin monoclonal antibody is using cyphenothrin hapten-carrier protein conjugate as immunogene
It prepares, is to be secreted to obtain by cyphenothrin monoclonal antibody hybridoma cell strain；The sheep anti mouse antiantibody is by mouse
Antibody mediated immunity sheep obtains in source.
The sample absorption pad (1), conjugate release pad (2), reaction film (3), water absorption pad (4) are successively pasted onto bottom plate
(7) on, the conjugate release pad 1/3~1/2 is capped under sample absorption pad.
The bottom plate can be the material that PVC bottom plate or other hard do not absorb water；The sample absorption pad can for suction strainer paper or
Filter paper for oil；The conjugate release pad can be mineral wool or polyester material；The water absorption pad is blotting paper；The reaction film can be
Nitrocellulose filter or cellulose acetate film.
It is a further object to provide a kind of methods for preparing above-mentioned test strips comprising step:
1) preparation is coated with cyphenothrin monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with cyphenothrin hapten-carrier protein conjugate and is coated with sheep anti mouse
The reaction film of the nature controlling line of antiantibody；
And 2) 3) 1) the conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate that prepare are assembled into
Test strips.
Specifically, step includes:
1) prepared by haptens: cyphenothrin and 1- formoxyl cyclopropane -2- carboxylic acid reaction are obtained cyphenothrin half
Antigen；
2) by cyphenothrin haptens and carrier protein couplet, cyphenothrin hapten-carrier albumen coupling is obtained
Object；
3) mouse is immunized with cyphenothrin hapten-carrier protein conjugate, by mouse boosting cell and myeloma cell
By merging, screening, cyphenothrin monoclonal hybridoma strain is obtained；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
6) the cyphenothrin monoclonal antibody by step 3) preparation is added in the colloidal gold of step 5) preparation, obtains phenylate
Cyano chrysanthemate monoclonal antibody-colloid gold label object；
7) cyphenothrin monoclonal antibody-colloid gold label object is sprayed in conjugate release pad, after 37 DEG C of baking 1h
It takes out, is placed in dry environment and saves backup；
8) cyphenothrin hapten-carrier protein conjugate is coated on reaction film and constitutes detection line, by sheep anti mouse
Antiantibody, which is coated on reaction film, constitutes nature controlling line；
9) sample absorption pad is used and contains 0.5% bovine serum albumin(BSA) (volume fraction), pH 7.2,0.1mol/L phosphate
Buffer impregnates 2h, dries 2h at 37 DEG C；
10) upper sample absorption pad, conjugate release pad, reaction film, water absorption pad are pasted in order on bottom plate, sample absorbs
Pad covers conjugate release pad, is finally cut into the small item of 3mm wide, adds plastic casing, is vacuum-packed, and can be reserved under the conditions of 4~30 DEG C
12 months.
It is a further object to provide phenylate cyanogen chrysanthemums in a kind of above-mentioned test strips detection vegetables of application and crops
The remaining method of ester, it comprising steps of
(1) sample pre-treatments；
(2) it is detected with test strips；
(3) analysis detection result.
Cyphenothrin Rapid detection test strip of the invention is pressed down using the antibody antigen reaction and competition of high degree of specificity
Cyphenothrin monoclonal antibody-colloid gold label object is fixed in conjugate release pad, sample by immunochromatographiassays assays technology processed
Cyphenothrin in product is in flow process, with cyphenothrin monoclonal antibody-colloidal gold mark in conjugate release pad
Remember that object combines, forms drug-antibody-colloid gold label object.The cyphenothrin on drug and reaction film detection line in sample
Hapten-carrier protein conjugate competitive binding cyphenothrin monoclonal antibody-colloid gold label object, according to detection line red
Band whether there is or not or shade judge in analyte sample fluid whether to remain containing cyphenothrin.
When detection, sample is instilled after processing in test strips hole, when the concentration of cyphenothrin in the sample is lower than detection
When limiting or being zero, monoclonal antibody-colloid gold label object meeting and the cyphenothrin being fixed on reaction film in chromatography process
Hapten-carrier protein conjugate combines, and a red stripes respectively occurs in detection line (T) and nature controlling line (C)；If phenylate
The concentration of Cyano chrysanthemate in the sample is equal to or higher than detection limit, and monoclonal antibody-colloid gold label object can be complete with cyphenothrin
Portion combines, thus because competitive reaction will not be in conjunction with cyphenothrin hapten-carrier protein conjugate without going out at T line
Existing red stripes.
Test strips of the invention have that high sensitivity, high specificity, at low cost, easy to operate, detection time is short, it is each to be suitble to
The advantages of kind unit uses, storage is simple, long shelf-life.With the remaining method letter of test strips of the present invention detection cyphenothrin
Just, quickly, intuitively, accurately, applied widely, at low cost, easy popularization and use.
Detailed description of the invention
Fig. 1 is cyphenothrin hapten synthesis figure.
Fig. 2 is test strips the schematic diagram of the section structure.
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 cyphenothrin test strip of embodiment
The preparation method of the test strips mainly comprises the steps that
1) preparation is coated with cyphenothrin monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with cyphenothrin hapten-carrier protein conjugate and is coated with sheep anti mouse
The reaction film of the nature controlling line of antiantibody；
And 2) 3) 1) conjugate release pad, reaction film and the sample absorption pad, water absorption pad and PVC bottom plate that prepare are assembled
At test strips.
Substep narration in detail below:
1, the preparation of cyphenothrin haptens
(1) cyphenothrin (compound a) 0.375g is taken, tetrahydrofuran 30ml is added to dissolve, 0-5 DEG C of stirring 20min adds hydrogen
Change aluminium lithium 0.11g, is restored to room temperature, is gradually heated up back flow reaction 4h.Stop reacting, water 30ml on the rocks, ethyl acetate 30ml ×
3, extraction three times, merges organic phase, is evaporated, and methylene chloride/n-hexane (v/v, 1/10) 80ml recrystallization obtains 0.34g product
Compound b, yield 91.89%.
(2) 0.34g compound b is taken, adds ethyl alcohol 50ml to dissolve, adds 1- formoxyl cyclopropane -2- carboxylic acid 0.12g, add three second
Amine 0.1ml, is stirred at room temperature 8h, is transferred to 0-5 DEG C of stirring 10min, adds sodium borohydride 0.11g, continues to stir 1h.Stop reaction,
Revolving is evaporated, and adds water 30ml, ethyl acetate 30ml × 3, and extraction three times, merges organic phase, is evaporated, upper silicagel column, and methylene chloride/
Methanol (v/v, 10/1) elution separation, obtains 0.38g haptens product Compound d, yield 88.37%.
2, the preparation of immunogene
20mg compound d is taken, adds 2ml DMF to dissolve, adds 17mg NHS, 23mg EDC that 1h is stirred at room temperature, obtains haptens
Activating solution A liquid, takes bovine serum albumin(BSA) 100mg, adds 0.05mol/L PB buffer solution, obtains B liquid, and A drop is added to B liquid
In, 4 DEG C are stirred overnight, 0.02mol/L PB buffer dialysis purification, obtain immunogene cyphenothrin-BSA, and -20 DEG C save,
It is spare.
3, the preparation of coating antigen
12mg compound d is taken, adds 2ml DMF to dissolve, adds 11mg NHS, 17mg EDC that 1h is stirred at room temperature, obtains haptens
Activating solution A liquid, takes ovalbumin 80mg, adds 0.05mol/L PB buffer solution, obtains B liquid, A drop is added in B liquid, 4
It DEG C is stirred overnight, 0.02mol/L PB buffer dialysis purification, obtains immunogene cyphenothrin-OVA, -20 DEG C of preservations are standby
With.
4, the preparation of cyphenothrin monoclonal antibody
(1) animal immune
In the immunogene injection Balb/c Mice Body that step 2 is obtained, immunizing dose is 150 μ g/, its is made to generate anti-blood
Clearly.
(2) cell fusion and cloning
Immune Balb/c mouse boosting cell is taken, is merged in 8:1 (quantitative proportion) ratio with SP2/0 myeloma cell, is used
Indirect competitive ELISA method measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, directly
To obtaining the hybridoma cell strain of stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 1 × 10 with frozen stock solution6The cell suspension of a/ml, saves for a long time in liquid nitrogen.When recovery
Cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, after centrifugation removal frozen stock solution, moves into culture culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, with octanoic acid-
Saturated ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture medium, and calf serum is made to exist
Final concentration of 20% (mass fraction) in cell culture medium, the final concentration of 0.2% (matter of sodium bicarbonate in cell culture medium
Measure score)；The pH of the cell culture medium is 7.4.
5, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, it is anti-to obtain sheep anti mouse
Antibody.
6, cyphenothrin monoclonal antibody-colloid gold label object preparation
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01% (mass fraction) with double distilled deionized water, 100ml is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring 1% trisodium citrate of 2.5ml, continue
Solution is at the uniform velocity heated with stirring in stopping when bright red, is restored to original volume, 4 DEG C of guarantors with deionized water after being cooled to room temperature
It deposits.The colloidal gold appearance prepared is pure, it is bright, without precipitating and floating material.
(2) cyphenothrin monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, molten by every milliliter of colloidal gold with the pH value of 0.2mol/L solution of potassium carbonate tune colloidal gold to 7.0
Cyphenothrin monoclonal antibody is added into colloidal gold solution for the standard that 20~50 μ g are added in liquid, continues to stir and evenly mix
10%BSA is added in 30min, makes its final concentration of 1% (volume fraction) in colloidal gold solution, stands 10min.12000r/
Min, 4 DEG C of centrifugation 40min abandon supernatant, and it is initial colloid gold volume 1/10 with volume that precipitating is washed twice with redissolution buffer
Redissolution buffer will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer: casein containing protein 0.02%~0.1% (mass fraction), 0.05%~0.2% (quality of Tween-80
Score), the 0.02mol/L phosphate buffer of pH7.2.
7, the preparation of conjugate release pad
Conjugate release pad is soaked in (concentration of the bovine serum albumin(BSA) in buffer is containing bovine serum albumin(BSA)
0.5%), in the phosphate buffer of pH 7.2,0.5mol/L, 1h is uniformly soaked, 37 DEG C of baking 3h are spare.Film is sprayed with Isoflow
By the cyphenothrin prepared monoclonal antibody-colloid gold label object even application in conjugate release pad, every 1cm is tied instrument
After closing object release pad spraying 0.01ml cyphenothrin monoclonal antibody-colloid gold label object, it is placed in (humidity < in 37 DEG C of environment
20%) it is taken out after 60min, is placed in dry environment (humidity < 20%) and saves backup.
8, the preparation of reaction film
Detection line will be constituted in cyphenothrin haptens-ovalbumin conjugate coating to reaction film, sheep anti mouse is resisted
Antibody, which is coated on reaction film, constitutes nature controlling line.
Coating process: being diluted to 10mg/ml for cyphenothrin haptens-ovalbumin conjugate with phosphate buffer,
It is coated in the detection line (T line) on nitrocellulose filter with Isoflow point film instrument, package amount is 0.8 μ l/cm；With
Sheep anti mouse antiantibody is diluted to 200 μ g/ml by the phosphate buffer of 0.01mol/L, pH7.4, with Isoflow point film instrument by its
The nature controlling line (C line) being coated on nitrocellulose filter, package amount are 1.0 μ l/cm.The reaction film being coated with is placed in 37 DEG C of items
Dry 2h, spare under part.
9, the preparation of sample absorption pad
Sample absorption pad is placed in slow containing 0.5% bovine serum albumin(BSA) (volume fraction), pH7.2,0.1mol/L phosphate
2h is impregnated in fliud flushing, 37 DEG C of baking 2h are spare.
10, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on PVC bottom plate in order；In conjunction with
Object release pad has 1/3 region to be absorbed by the sample pad covering from starting point, and the end of conjugate release pad and the beginning of reaction film connect
It connects, the end of reaction film is connected with the beginning of water absorption pad, and the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, water absorption pad
End be aligned with the end of PVC bottom plate；There are detection line and nature controlling line, detection line (T line) and nature controlling line (C on the reaction film
Line) it is the strip tape perpendicular with the length of the test strips；Detection line is located at the side close to the end of conjugate release pad；
Nature controlling line is located remotely from the side of the end of conjugate release pad；Test strips are cut into the small item of 3mm wide with machine, mounted in special
Plastics fabrication in, under the conditions of 4~30 DEG C can be reserved for 12 months.
The remaining detection of cyphenothrin in 2 sample of embodiment
1, it is detected with test strips
Measuring samples solution, which to be drawn, with suction pipe 3 drops is vertically added dropwise in well, liquid starts timing when flowing, reaction 5~
10min determines result.
2, analysis detection result
Result is read by colloidal gold analyzer (referred to as " analyzer "):
Negative (-): indicate that test substance concentration is lower than detection limit in sample；
Positive (+): indicate that test substance concentration is equal to or higher than detection limit in sample；
It is invalid: to indicate to need to retest.
3 sample detection example of embodiment
1, detection limit test
Take blank Chinese cabbage and apple sample, wherein respectively addition cyphenothrin to final concentration of 0.5,1,2 μ g/kg,
Test strips are taken to be detected, each sample is repeated three times.
When detecting Chinese cabbage and apple sample with test strips, when wherein cyphenothrin addition concentration is 0.5 μ g/kg, point
Analyzer is shown as negative；When wherein cyphenothrin addition concentration is 1,2 μ g/kg, analyzer is shown as positive.
2, false positive rate, false negative rate test
Take the Chinese cabbage and each 20 parts of apple positive sample and content that known cyphenothrin content is 1 μ g/kg less than 0.5 μ
The Chinese cabbage of g/kg and each 20 parts of apple negative sample, are detected with three batches of test strips, calculate its yin and yang attribute rate.It the results are shown in Table 1,
Table 2.
1 Chinese cabbage of table detects sample results
2 apple of table detects sample results
The result shows that: when detecting positive Chinese cabbage and apple sample with the test strips of 3 batch productions, as a result it is all positive,
Know that positive sample coincidence rate is 100%, false negative rate 0；When detecting 20 parts of negative Chinese cabbages and apple sample, it is as a result all yin
Property, it is known that negative match-rate 100%, false positive rate 0.Illustrate that the test strips of detection cyphenothrin of the invention can be right
Cyphenothrin residual is used for quickly detecting in Chinese cabbage and apple.
3, specific test
The drugs such as 500 μ g/kg cypermethrins, fenvalerate and decis are detected with cyphenothrin test strips.As a result
It has been shown that, test strips nature controlling line and detection line develop the color, are negative.Illustrate this test strips to 500 μ g/kg cypermethrins, penta chrysanthemum of cyanogen
The drugs no cross reaction such as ester and decis.
Claims (8)
1. it is a kind of detect cyphenothrin test strips, including sample absorption pad (1), conjugate release pad (2), reaction film (3),
Water absorption pad (4) and bottom plate (7), it is characterised in that have on the reaction film and be coated with cyphenothrin hapten-carrier albumen
The detection line (5) of conjugate and the nature controlling line (6) for being coated with sheep anti mouse antiantibody, the conjugate release pad (2) are coated with benzene
Ether Cyano chrysanthemate monoclonal antibody-colloid gold label object, the cyphenothrin haptens the preparation method is as follows:
(1) cyphenothrin (compound a) 0.375g is taken, tetrahydrofuran 30ml is added to dissolve, 0-5 DEG C of stirring 20min, plus hydrogenated aluminium
Lithium 0.11g, is restored to room temperature, is gradually heated up back flow reaction 4h；Stop reaction, water 30ml on the rocks, ethyl acetate 30ml × 3, extraction
It takes three times, merges organic phase, be evaporated, methylene chloride/n-hexane (v/v, 1/10) 80ml recrystallization obtains 0.34g product chemical combination
Object b, yield 91.89%；
(2) 0.34g compound b is taken, adds ethyl alcohol 50ml to dissolve, adds 1- formoxyl cyclopropane -2- carboxylic acid 0.12g, add triethylamine
8h is stirred at room temperature in 0.1ml, is transferred to 0-5 DEG C of stirring 10min, adds sodium borohydride 0.11g, continues to stir 1h；Stop reaction, rotation
Steaming is evaporated, and adds water 30ml, ethyl acetate 30ml × 3, and extraction three times, merges organic phase, is evaporated, upper silicagel column, methylene chloride/first
Alcohol (v/v, 10/1) elution separation, obtains 0.38g haptens product Compound d, yield 88.37%.
2. test strips as described in claim 1, it is characterised in that the sample absorption pad (1), conjugate release pad (2), anti-
Film (3), water absorption pad (4) is answered successively to be pasted on bottom plate (7).
3. such as the described in any item test strips of claim 1-2, it is characterised in that the conjugate release pad 1/3~1/2 is coating
It is placed under sample absorption pad.
4. test strips as described in claim 1, it is characterised in that the cyphenothrin hapten-carrier protein conjugate by
Cyphenothrin haptens is obtained with carrier protein couplet, and the carrier protein is bovine serum albumin(BSA), ovalbumin, blood indigo plant egg
White, thyroprotein, human serum albumins.
5. such as the described in any item test strips of claim 1 or 4, it is characterised in that the cyphenothrin haptens is by phenylate
Cyano chrysanthemate is obtained with 1- formoxyl cyclopropane -2- carboxylic acid reaction, molecular structural formula are as follows:
6. test strips as described in claim 1, it is characterised in that the cyphenothrin monoclonal antibody is with phenylate cyanogen chrysanthemum
Ester hapten-carrier protein couplet object is prepared as immunogene, and the sheep anti mouse antiantibody is by source of mouse antibody mediated immunity sheep
It obtains.
7. a kind of method for preparing any one of claim 1-6 test strips comprising step:
1) preparation is coated with cyphenothrin monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with cyphenothrin hapten-carrier protein conjugate and is coated with sheep anti mouse and resists
The reaction film of the nature controlling line of body；
And 2) 3) 1) the conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate that prepare are assembled into test paper
Item.
8. the remaining method of cyphenothrin in a kind of crops such as detection veterinary antibiotics comprising step:
1) Sample pretreatment；
2) it is detected with test strips described in any one of claims 1-6；
3) analysis detection result.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811079749.4A CN109682961B (en) | 2018-09-17 | 2018-09-17 | Test strip for detecting cyphenothrin and application thereof |
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|---|---|---|---|
| CN201811079749.4A CN109682961B (en) | 2018-09-17 | 2018-09-17 | Test strip for detecting cyphenothrin and application thereof |
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| CN109682961B CN109682961B (en) | 2022-09-20 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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