CN108982843A - A kind of test strips and preparation method thereof of effective cypermethrin pesticide quantitative detection - Google Patents
A kind of test strips and preparation method thereof of effective cypermethrin pesticide quantitative detection Download PDFInfo
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Abstract
The invention discloses a kind of test strips and preparation method thereof of effective cypermethrin pesticide quantitative detection.It should include carrier mat, sample pad, bonding pad, nitrocellulose filter and water absorption pad；Sample pad, bonding pad, nitrocellulose filter and water absorption pad are successively closely coupled and are pasted onto carrier mat upper surface, wherein the effective cypermethrin antibody of lanthanide label is attached on bonding pad；Detection line and nature controlling line are provided on nitrocellulose filter；Bonding pad, detection line, nature controlling line and water absorption pad are arranged successively；Effective cypermethrin envelope antigen and secondary antibody are respectively fixed in detection line and nature controlling line.The present invention utilizes the chelate labels pyrethroids antibody of lanthanide, and the test strips holding time of acquisition is long, low in cost, is able to achieve the fast quantitative analysis to pyrethrin pesticide, and high sensitivity, and detection limit can achieve 1ng/ml.
Description
Technical field
The invention belongs to effective cypermethrin field of pesticide detection, in particular to a kind of effective cypermethrin pesticide is quantitatively examined
Test strips of survey and preparation method thereof.
Background technique
" bread is the staff of life, eats with An Weixian ".Food is the material base that the mankind depend on for existence with development, and food safety is asked
Topic is related to the health and safety of the people, is the significant problem of national economy.But with the lasting height of economy and society
Speed development, while solving the problems, such as safety (the food security) of quantity of food substantially, the safety (food of food matter
Safety) problem increasingly causes the concern of the whole society, and the negative news in relation to food safety also emerges one after another.Countries in the world are just
It is faced with severe food safety challenge, food origin disease is mainly manifested in and persistently rises, pernicious food pollution event occurs frequently not
Thoroughly, food production or New Machining Technology, new process bring new harm, and world wide is interior due to food safety and sanitation quality problems
Caused by food trade dispute it is continuous, affect between various countries economic trade development.
Now, although the agricultural avocation production of countries in the world has large development, per unit area yield is low, poor quality, agricultural and sideline product agriculture
Medicine residual is exceeded, certain to have forbidden the pesticide used in production still and have to be used, and causes " 3R " problem (i.e. pesticide residue
Residue, pest resistance to insecticide Reistance, Pest Resurgence Resurgence) it is more and more prominent.Pesticide residue (abbreviation agriculture
It is residual) problem is most important safety problem in agricultural avocation production process.In the planting process of crops, people are increasingly dependent on
Dazzling chemical insecticide, chemical herbicide, antibiotic and efficient chemical fertilizer etc..The residual exceeded production of agriculture is eaten for a long time
Product, even if can also cause the slow poisoning of humans and animals without result in acute poisoning, generation or even shadow so as to cause disease
It rings and arrives descendants.
Effective cypermethrin class pesticide is in synthetic pesticide insecticide development history after organochlorine, organic phosphorus, carbamate
It is carried out on the basis of the analoglike pyrethrum flower effective ingredient developed the 1970s by offshore company after pesticide
Artificial synthesized bionic pesticide, its exploitation are considered the third milestone in Agrochemicals history.Since it is living with desinsection
Property it is high, dosage is few, residual quantity is low, to mammal small toxicity the characteristics of, be that country advocates the Pesticidal products promoted.As having
The substitute products of machine phosphorus insecticide, usage amount increase year by year, are widely used in the fields such as agricultural, forestry, hygienic industry.It is mainly used
Pest harmful mite on the crops such as prevention and treatment cotton, vegetables, fruit tree, tea tree, tobacco, soybean can effectively prevent coleoptera, half
Wing mesh, Homoptera and lepidoptera pest and harmful mite, such as bollworm, anthonomus grandis, alfalfa weevil, looper, smaller green leaf hopper, apple
Stupid moth, diamondback moth, cabbage caterpillar, Lay white butterfly, America mythimna separata, colorado potato bug, cutworm, corn borer, aleyrodid, aphid, spider mite kind,
The 140 various pests harmful mite such as goitre mite class.Simultaneously, it may also be used for prevent and treat the domestic hygienes pests such as mosquitos and flies and fish with insecticidal materials in water
It produces and is applied in cultivation.But due to long-term a large amount of uses of pyrethrin pesticide, also various pests harmful mite can be made to develop drug resistance, and
Fish, honeybee and natural enemy can be generated compared with high toxicity.Pyrethrin pesticide can threaten people by food chain in the intracorporal residual of fish
Body health.Expert's discovery, fenvalerate may have environmental estrogenic activity, and 30 μm of ol/L fenvalerates can make one endometrium
Cancer Ishikawa Var-I and breast cancer T47D cell line abnormality proliferation, can make one breast cancer mcf-7 cell line abnormality proliferation.Chrysanthemum
Ester pesticides, which are absorbed or taken orally by human skin for a long time, can cause to be poisoned, and can lead to flesh by influencing the conduction of neural axon
Meat spasm etc..
Pesticide Residue is paid much attention in countries in the world, residual to the agriculture in various agricultural and sideline products all to formulate harsh limitation
Standard, so that agricultural and sideline product outlet in China's is faced with formidable challenges.For the rapid detection method of pesticide residue, it is each to become the world
State's primary study object.Gas-chromatography or gas chromatography mass spectrometry chromatography mainly are passed through to the quantitative detection of pyrethrin pesticide at present, by
What professional technician completed in laboratory.This detection method needs the time long, expensive, is unable to satisfy real work
Needs.For simply and easily detect agricultural product pyrethrin pesticide presence, Chinese patent application CN102507931A mentions
A kind of pyrethrin pesticide colloidal gold test has been supplied, has been by carrier board, nitrocellulose membrane, colloidal gold pad, sample-adding pad and to inhale
Pad composition is received, carrier board is located at bottom, and nitrocellulose membrane, colloidal gold pad, sample-adding pad, absorption pad are successively pasted onto table on carrier board
Face, nitrocellulose membrane are located in the middle part of carrier board, colloidal gold pad be located at the side of nitrocellulose membrane and with cellulose nitrate membrane part weight
Folded, sample-adding pad is located in colloidal gold pad and partly overlaps with colloidal gold pad, absorption pad be located at the other side of nitrocellulose membrane and with
Nitrocellulose membrane partly overlaps, and one layer is uniformly coated in colloidal gold pad the anti-effective cypermethrin Dan Ke of the mouse of colloid gold label
Grand antibody is equipped with the detection line for being coated with detection antigen on nitrocellulose membrane close to sample-adding pad one end, sets close to absorption pad one end
There is the nature controlling line for being coated with Quality Control antibody.This test paper is although easy to use, but it exists simultaneously following point: (1) product
Detection limit it is higher, be unable to satisfy the limitation requirement in agricultural product to pyrethrin pesticide；(2) quantitative effect of colloidal gold test
It is poor, quantitative detection can not be carried out to the pyrethroids in agricultural product.
Determining pyrethroid pesticide residues mainly pass through gas-chromatography and are detected with gas chromatography mass spectrometry chromatography at present, these methods
To sample pre-treatments very complicated, height is required to operating technology, and instrument and equipment is expensive, need technical professional's progress
The disadvantages of operation, it is long that there are detection times, and testing cost is expensive.Although there is exploitation colloidal gold immunity chromatography that can apply at present
In the quick detection of Determining pyrethroid pesticide residues, but colloidal gold immunity chromatography is higher to pyrethrin pesticide minimum detection limit,
And it can not carry out accurately quantifying, be unable to satisfy the requirement of Limited Doses in agricultural product.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of effective cypermethrin pesticide
The test strips of quantitative detection.The test strips are used cooperatively with Portable detection instrument and are able to achieve to the fast of effective cypermethrin class pesticide
Speed is quantitative, and detection limit can achieve 1ng/ml, is current most sensitive fast quantification effective cypermethrin detection technique.
Another object of the present invention is to provide the preparation sides of the test strips of the effective cypermethrin pesticide quantitative detection
Method.
The purpose of the invention is achieved by the following technical solution: a kind of test paper of effective cypermethrin pesticide quantitative detection
Item, including carrier mat, sample pad, bonding pad, nitrocellulose filter and water absorption pad；Sample pad, bonding pad, nitrocellulose filter and
Water absorption pad is successively closely coupled and is pasted onto carrier mat upper surface, wherein the efficient of lanthanide label is attached on bonding pad
Cypermethrin antibody；Detection line (T line) and nature controlling line (C line) are provided on nitrocellulose filter；Bonding pad, detection line, Quality Control
Line and water absorption pad are arranged successively；Effective cypermethrin envelope antigen and secondary antibody are respectively fixed in detection line and nature controlling line.
The sample pad and bonding pad is combined into sample combination pad, is covered with glue above the sample segment combination mat
Belt, adhesive tape layer prevent dropwise addition sample excessive, and sample covers the effect of nitrocellulose membrane.
The lanthanide is preferably europium (Eu)；Europium reagent is easy to get, and fluorescent value is stablized after being excited, and is more advantageous to
The stability of product of the present invention.
The effective cypermethrin antibody of the lanthanide label is prepared preferably by following method: will be efficient
Cypermethrin antibody and DTTA-Eu3+Ambient temperature overnight after labelled reagent mixes, the effective cypermethrin for obtaining lanthanide label are anti-
Body.
The effective cypermethrin antibody and DTTA-Eu3+The mass ratio of labelled reagent is preferably 5:1.
The effective cypermethrin antibody is prepared via a method which to obtain:
(1) mouse is immunized with effective cypermethrin artificial antigen, the mouse boosting cell after being immunized is taken to melt with myeloma cell
Culture is closed, screening positive hybridoma cell carries out in vitro culture, and clone freezes after expanding culture, obtains hybridoma；
(2) mouse peritoneal injection is carried out with norphytane, be inoculated into hybridoma obtained in step (1) after 6 days small
In mouse abdominal cavity, ascites is collected after 5 days, and ascites is purified, obtain effective cypermethrin antibody.
Effective cypermethrin artificial antigen described in step (1) is prepared preferably by following method:
1. the preparation of effective cypermethrin haptens: by isophthalic oxygen methyl benzoic acid 21.2mg and 11.4mg NHS (N- hydroxyl
Base succinimide) be dissolved in 2ml DMF (dimethylformamide), add 20.6mg DCC (dicyclohexylcarbodiimide) into
Row reaction, obtains the haptens of effective cypermethrin；
2. the preparation of effective cypermethrin artificial antigen: by step 1. obtained in effective cypermethrin haptens with
KLH (keyhole limpet hemocyanin) carries out coupling reaction, obtains effective cypermethrin artificial antigen.
Step 1. described in time of reaction be preferably 4 hours.
Mouse described in step (2) is preferably BALB/c mouse.
Purifying described in step (2) is purified preferably by saturated ammonium sulfate method；More preferably as follows
It realizes:
(I) mouse ascites are centrifuged, collect supernatant, after obtained mouse supernatant is mixed with isometric physiological saline, drop
Add saturated ammonium sulfate solution, be then centrifuged for making albumen precipitation, is then centrifuged for, abandons precipitating；
(II) continue that saturated ammonium sulfate solution is added dropwise into supernatant obtained in step (1), stand, centrifugation, abandon supernatant,
Obtain precipitate B；
(III) it will be dialysed after precipitate B physiological saline solution with PBS solution.
The condition of centrifugation described in step (I) is preferred are as follows: 4 DEG C or at room temperature, 13000r/min be centrifuged 30min.
The volume ratio of mouse supernatant described in step (I) and saturated ammonium sulfate solution is 1:1.
Standing described in step (I) is 4 DEG C of standing 1h.
Centrifugation described in step (II) is to be centrifuged under the conditions of 4 DEG C.
The volume ratio of supernatant described in step (II) and saturated ammonium sulfate solution is 1:0.5.
Dialysis described in step (III) is to be dialysed with bag filter.
The temperature of the dialysis is preferably 4 DEG C.
The effective cypermethrin envelope antigen is prepared via a method which to obtain:
(a) preparation of effective cypermethrin haptens: by isophthalic oxygen methyl benzoic acid and NHS (n-hydroxysuccinimide)
It is dissolved in DMF (dimethylformamide), adds DCC (dicyclohexylcarbodiimide) and reacted, obtain effective cypermethrin
Haptens；
(b) preparation of envelope antigen: the haptens of effective cypermethrin obtained in step (a) and BSA are reacted,
Obtain effective cypermethrin envelope antigen.
The secondary antibody is preferably sheep anti mouse secondary antibody.
The preparation method of the test strips of the effective cypermethrin pesticide quantitative detection, includes the following steps:
(i) the effective cypermethrin antibody that lanthanide marks is dispersed on bonding pad；
(ii) effective cypermethrin envelope antigen and sheep anti mouse secondary antibody are equably drawn in cellulose nitrate respectively with stroke film instrument
On plain film, detection line and nature controlling line are formed, then be dried；
(iii) nitrocellulose filter for obtaining the bonding pad that sample pad, step (i) obtain, step (ii) in carrier mat
It is successively mutually overlapped with water absorption pad, wherein for the detection line of nitrocellulose filter close to bonding pad, nature controlling line is close far from bonding pad
Water absorption pad obtains the test strips of effective cypermethrin pesticide quantitative detection.
Drying described in step (ii) preferably at 25 DEG C of room temperature, relative humidity less than 40% under conditions of done
It is dry.
It carries out drawing the concentration of film being preferably 1mg/mL with stroke film instrument described in step (ii).
Detection line described in step (ii) and the spacing of nature controlling line are preferably 0.39cm.
Application of the test strips of the effective cypermethrin pesticide quantitative detection in detection field.
The action principle of test paper of the present invention: marking effective cypermethrin antibody by europium (Eu), under the action of blotting paper,
By capillarity, the antibody of label is chromatographed along nitrocellulose membrane, with the pyrethroids envelope antigen (detection on nitrocellulose membrane
Line) and secondary antibody (nature controlling line) combination, the time difference fluorescence intensity ratio of detection line and nature controlling line is read according to portable instrument, quickly
Effective cypermethrin concentration in quantitative analysis sample.
The present invention has the following advantages and effects with respect to the prior art:
1, time-resolved fluorescence technology (time-resolved fluorescence, abbreviation TRF) is based on Rare Earth Lanthanum race
Element such as Eu, dysprosium (Dy), samarium (Sm) etc. have the characteristics that the on-radiation detection technique that longer fluorescence lifetime develops.This hair
The bright fluorescence intensity using two kinds of fluorescence measurement techniques measurement final products of time resolution and wavelength resolution, according to fluorescence intensity and phase
To fluorescence intensity ratio, the concentration of analyte in reaction system is determined.The UV Absorption coordination that Rare Earth Lanthanum race ion is suitable for
Body chelating formed chelating object, by the light sources such as ultraviolet light, xenon lamp excitation and emit fluorescence.TRF technology, which effectively eliminates, is
The non-specific fluorescence signals such as system background fluorescence, have hypersensitive, quantitative analysis wide dynamic range, tracer stability good, not dirty
Outstanding advantages of environment and application is wide is contaminated, many advantages of TRF technology are fundamentally consolidating from lanthanide complex
There is feature.
2, test strips produced by the present invention utilize the chelate of lanthanide europium (Eu) by the labeling method of improvement antibody
Pyrethroids antibody is marked, the fluorescence lifetime of rare earth metal Eu is longer, the analysis method of the fluorescence intensity detected after closing exciting light,
According to the ratio of the fluorescence intensity of reactor product and relative intensity of fluorescence, the pyrethroids concentration in reaction substrate is quickly detected.
The detection limit of product can improve nearly 20 times than colloidal gold test detection limit in CN102507931A, reach 1ng/ml, pass through
Specific Time-resolved fluorescence assay instrument, can be to the pyrethroids progress fast quantification inspection of 1ng/ml~100ng/ml in substrate
It surveys.
3, test paper product of the invention is easy to carry, can circulate under room temperature, and the holding time is long, low in cost, can combine
Associated portable instrument realizes that the fast quantitative analysis to pyrethrin pesticide, product can be applied to detection of agricultural products mechanism and one
A little bases monitor unit, realize the safe and effective inspection with chrysanthemum esters medicine to agricultural product.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of test strips；Wherein, 1 is sample combination pad, and 2 be adhesive tape layer, and 3 be detection line, and 4 be nitre
Acid cellulose film, 5 be nature controlling line, and 6 be water absorption pad, and 7 be carrier mat.
Fig. 2 is the fluorescent value analysis chart of various concentration standard items C line Yu T line.
Fig. 3 is four parameter curve fit graph of equation of standard concentration Yu T line fluorescent value and C line fluorescence ratio (T/C).
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
The present invention is by being uniformly sprayed on sample combination pad, in nitrocellulose membrane with after lanthanide Eu labelled antibody
Quality Control line position draws upper secondary antibody, draws upper envelope antigen in the detection line position of nitrocellulose membrane, is assembled into quick detection test paper.
It is prepared as follows:
(1) preparation of effective cypermethrin haptens:
Taking 21.2mg isophthalic oxygen methyl benzoic acid (being purchased from sigma) and 11.4mg NHS, (n-hydroxysuccinimide is purchased from
Sigma) in the centrifuge tube of 5mL, 2.0mL DMF (dimethylformamide is purchased from sigma) mixing is added.Take 20.6mg
DCC (dicyclohexylcarbodiimide is purchased from sigma) is dissolved in 1ml DMF.Hybrid reaction 5h at room temperature obtains efficient chlorine
The haptens of Cyano chrysanthemate.
(2) preparation of the immunizing antigen and envelope antigen of effective cypermethrin: taking above-mentioned 10 μ l of haptens, and KLH (key is added
Hole relative hemocyanin, 20mg/ml) 100 μ l and PBS (0.01M) 800 μ l, react 1 day at room temperature, are prepared into immunizing antigen, are used for
Immune balb/c mice (6 week old BALB/C mices, Shanghai Experimental Animal Center) uses.Above-mentioned 10 μ l of haptens is taken, BSA is added
(bovine serum albumin(BSA), 20mg/ml) 100 μ l and PBS (0.01M) 800 μ l reacts 1 day at room temperature, is prepared into envelope antigen, uses
Film is drawn in chromatography reagent strip detection line.
(3) mouse (6 week old the preparation of effective cypermethrin monoclonal antibody: are immunized with effective cypermethrin artificial antigen
BALB/C mice, Shanghai Experimental Animal Center), (Xiamen University cures by the spleen cell for taking immune mouse and SP2/0 myeloma cell
Institute) fusion culture, positive hybridoma cell in vitro culture is screened, clone freezes (freezing temperature is -70 DEG C) after expanding culture,
BALB/c mouse or its parent mouse are selected, first carries out mouse peritoneal injection with norphytane 1ml (the raw work in Shanghai), it will be miscellaneous after 6 days
It hands over oncocyte to be inoculated into mouse peritoneal, then collects ascites after 5 days.
(4) purifying of effective cypermethrin monoclonal antibody: saturated ammonium sulfate method is referred to, the specific steps are as follows: above-mentioned pumping
The mouse ascites taken collect supernatant through 4 DEG C or room temperature centrifugation (13000r/min, 30min)；Take honest and upright and thrifty 20ml and isometric life
After managing salt water mixing, 40ml saturated ammonium sulfate solution (SAS) is slowly added dropwise with stirring, in precipitating 30min or more or overnight, makes
Albumen sufficiently precipitates；4 DEG C, 13 000r/min centrifugation 10min, abandon supernatant；It is precipitated with 12ml physiological saline solution, 8ml is added dropwise
SAS, 4 DEG C of standing 1h, 4 DEG C of centrifugations are abandoned supernatant, are precipitated with 13.3ml physiological saline solution.6.6ml SAS, 4 DEG C of standings are added dropwise
Supernatant is abandoned in 1h, 4 DEG C of centrifugations, and centrifuged pellet object is dissolved with a little 0.01M PBS to be precipitated, and is packed into bag filter；With big
It dialyses in the PBS solution (concentration 0.01M, pH 7.4) of 20 times of volumes in 4 DEG C, replacement dialyzate for several times, sets -20 DEG C of preservations.
(5)Eu3+The preparation of effective cypermethrin antibody: (efficient chlorine cyanogen is obtained in step (4) according to label 1mg conjugate
Pyrethroids monoclonal antibody) need 0.2mg DTTA-Eu3+(isothiocycmatobenzyl diethylenetriamines tetraacethyl europium is purchased from PE company)
Labelled reagent, i.e. mass ratio are 5:1, by antigen-antibody to be marked and the sufficiently mixed hook of labelled reagent, are placed in ambient temperature overnight.
(6) it the processing of nitrocellulose filter: uses and draws film instrument for effective cypermethrin envelope antigen and sheep anti mouse secondary antibody
(sigma) it equably draws among nitrocellulose filter 4, at 25 DEG C of room temperature, relative humidity is done under the atmosphere less than 40%
It is dry.Effective cypermethrin envelope antigen forms detection line (T line) 3, and sheep anti mouse secondary antibody forms nature controlling line (C line) 5, wherein coating is anti-
Former and sheep anti mouse secondary antibody film concentration of drawing is 1mg/mL, draws film speed 50cm/min, every 1 μ l of cm dosage.And detection line 3 and matter
The spacing for controlling line 5 is 0.39cm.
(7) assembling of test strips: as shown in Figure 1, by carrier mat 7, nitrocellulose filter 4, sample combination pad 1, adhesive tape layer 2
And water absorption pad 6 is spliced into layer structure, carrier mat 7 is located at bottom, and the top of carrier mat 7 is equipped with nitrocellulose filter 4, sample
Combination mat 1 and water absorption pad 6 are respectively arranged at the both ends of nitrocellulose filter 4, and sample combination pad 1 and water absorption pad 6 respectively with nitre
Acid cellulose film 4 partly overlaps, and the top of sample segment combination mat 1 is covered with adhesive tape layer 2, the detection on nitrocellulose filter 4
Line 3 is close to one end of sample combination pad 1, and nature controlling line 5 is close to one end of water absorption pad 6.Adhesive tape layer (common adhesive waterproof tape) prevents
Dropwise addition sample is excessive, and sample covers the effect of nitrocellulose membrane.
(8) the effective cypermethrin standard solution that 10ng/ml~320ng/ml various concentration is prepared with PBS solution, takes 3
It is added drop-wise to the adding mouth (in sample pad) of Test paper, is detected after 10min with JY1501FS fluorescent quantitation instrument fluorescence detector
After (Fig. 2), obtains the ratio of the fluorescence intensity of detection line and nature controlling line and standard concentration is four parameter fitting standard curves, R
Value is 0.99383, illustrates that the detection is stuck in the concentration range of 1ng/ml to 100ng/ml, relative intensity of fluorescence and sample it is dense
It spends line and closes correlation, can satisfy detection demand (Fig. 3).Wherein, it result judgement: is read according to the fluorescence chart scanner of standard items correction
Testing result, if illustrating that test paper is invalid without fluorescent value.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of test strips of effective cypermethrin pesticide quantitative detection, it is characterised in that: including carrier mat, sample pad, combination
Pad, nitrocellulose filter and water absorption pad；Sample pad, bonding pad, nitrocellulose filter and water absorption pad are successively closely coupled and paste
In carrier mat upper surface, wherein be attached with the effective cypermethrin antibody of lanthanide label on bonding pad；Nitrocellulose filter
On be provided with detection line and nature controlling line；Bonding pad, detection line, nature controlling line and water absorption pad are arranged successively；In detection line and nature controlling line
It is respectively fixed with effective cypermethrin envelope antigen and secondary antibody.
2. the test strips of effective cypermethrin pesticide quantitative detection according to claim 1, it is characterised in that: the lanthanum
Race's element is europium.
3. the test strips of effective cypermethrin pesticide quantitative detection according to claim 1, it is characterised in that: described two
Resist for sheep anti mouse secondary antibody.
4. the test strips of effective cypermethrin pesticide quantitative detection according to claim 1, which is characterized in that the lanthanum
The effective cypermethrin antibody of race's rubidium marking is prepared via a method which to obtain: by effective cypermethrin antibody and DTTA-Eu3+
Ambient temperature overnight after labelled reagent mixes obtains the effective cypermethrin antibody of lanthanide label.
5. the test strips of effective cypermethrin pesticide quantitative detection according to claim 4, it is characterised in that: the height
Imitate cypermethrin antibody and DTTA-Eu3+The mass ratio of labelled reagent is 5:1.
6. the test strips of effective cypermethrin pesticide quantitative detection according to claim 4, which is characterized in that the height
Effect cypermethrin antibody is prepared via a method which to obtain:
(1) mouse is immunized with effective cypermethrin artificial antigen, the mouse boosting cell after being immunized is taken to merge training with myeloma cell
It supports, screening positive hybridoma cell carries out in vitro culture, and clone obtains hybridoma after expanding culture；
(2) mouse peritoneal injection is carried out with norphytane, hybridoma obtained in step (1) is inoculated into mouse abdomen after 6 days
In chamber, ascites is collected after 5 days, and ascites is purified, obtain effective cypermethrin antibody；
Wherein, effective cypermethrin artificial antigen described in step (1) is prepared via a method which to obtain:
1. the preparation of effective cypermethrin haptens: isophthalic oxygen methyl benzoic acid 21.2mg and 11.4mg NHS are dissolved in 2ml
In DMF, adds 20.6mg DCC and reacted, obtain the haptens of effective cypermethrin；
2. the preparation of effective cypermethrin artificial antigen: by step 1. obtained in effective cypermethrin haptens and KLH into
Row coupling reaction obtains effective cypermethrin artificial antigen.
7. the test strips of effective cypermethrin pesticide quantitative detection according to claim 1, which is characterized in that the height
Effect cypermethrin envelope antigen is prepared via a method which to obtain:
(a) preparation of effective cypermethrin haptens: isophthalic oxygen methyl benzoic acid and NHS (n-hydroxysuccinimide) are dissolved in
It in DMF, adds DCC and is reacted, obtain the haptens of effective cypermethrin；
(b) preparation of envelope antigen: the haptens of effective cypermethrin obtained in step (a) and BSA are reacted, obtained
Effective cypermethrin envelope antigen.
8. the preparation method of the test strips of the described in any item effective cypermethrin pesticide quantitative detections of claim 1~7, special
Sign is, includes the following steps:
(i) the effective cypermethrin antibody that lanthanide marks is dispersed on bonding pad；
(ii) effective cypermethrin envelope antigen and sheep anti mouse secondary antibody are equably drawn in nitrocellulose filter respectively with stroke film instrument
On, detection line and nature controlling line are formed, then be dried；
(iii) nitrocellulose filter obtained the bonding pad that sample pad, step (i) obtain, step (ii) in carrier mat and suction
Water cushion successively mutually overlaps, and wherein the detection line of nitrocellulose filter is close to bonding pad, and nature controlling line is far from bonding pad, close to water suction
Pad, obtains the test strips of effective cypermethrin pesticide quantitative detection.
9. the preparation method of the test strips of effective cypermethrin pesticide quantitative detection according to claim 8, feature exist
In:
It carries out drawing the concentration of film being 1mg/mL with stroke film instrument described in step (ii).
10. the test strips of the described in any item effective cypermethrin pesticide quantitative detections of claim 1~7 are in detection field
Using.
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