CN108226489A - A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof - Google Patents
A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof Download PDFInfo
- Publication number
- CN108226489A CN108226489A CN201810005478.1A CN201810005478A CN108226489A CN 108226489 A CN108226489 A CN 108226489A CN 201810005478 A CN201810005478 A CN 201810005478A CN 108226489 A CN108226489 A CN 108226489A
- Authority
- CN
- China
- Prior art keywords
- ethiprole
- layer
- antibody
- preparation
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of colloid gold label test strips for quickly detecting micro ethiprole and preparation method thereof; the test strips bottom is supporting layer; middle layer is adsorption layer; protective layer is fixed on adsorption layer; adsorption layer is followed successively by the absorbent material layer of adsorbing fiber layer, gold labeling antibody fibrous layer, cellulose film layer and handle end from test lead; the carrier protein of coupling ethiprole is wherein coated on cellulose membrane as detection line, is additionally provided with the nature controlling line with goat-anti or rabbit anti-mouse IgG, goat anti-rabbit igg antibody or SPA printings.Experiments have shown that this test paper has stronger specific and higher sensitivity, detection is limited to 10ng/mL, with other pesticides or veterinary drug no cross reaction.
Description
Technical field
The present invention relates to a kind of utensils for quickly detecting micro insecticide drug, are quickly detected more particularly to one kind micro
Colloid gold label test strip of ethiprole and preparation method thereof.
Background technology
Ethiprole, trade name Frontline, english common name fipronil (fipronil), chemical name (RS) -5- ammonia
Base -1- (2,6- bis- chloro- a, a, a- trifluoro-p-tolyls) -4- trifluoromethyl sulfinyl pyrazole -3- nitriles, molecular formula are
C8N2Cl4, molecular formula C12H4Cl2F6N4OS, molecular weight 437.2 is Rhone-Poulenc Company's phenyl developed in 1981
Insecticidal pyrazolines.Begin to use from the mid-90 in last century, be widely used broad spectrum pesticide in a kind of world wide
And veterinary drug, it is mainly used for crop pests control, the treatment and control of house pet parasite, public health public place of entertainment pest
The various aspects such as prevention and control are used widely.It effectively inhibits insect central nervous system by being combined with GABA receptor
The chloride channel that γ-aminobutyric acid is adjusted in system, and have very high affinity with this channel, so as to interfere polypide maincenter
Nervous system causes its nerve and muscle surexcitation excited and dead.A large amount of result of the test shows the fluorine worm of various concentration
Nitrile aaerosol solution, pulvis or granule by foliar spray, seed pelleting, mix soil spread fertilizer over the fields etc. modes to rice grub such as snout moth's larva,
Brown paddy plant hopper, small brown rice planthopper and rice water weevil function and effect are good.Prevent effect also especially good pasture locust, control rate up to 73% with
On.To mosquito, meadow red fire ant also has particularly preferred control effect.Ethiprole is in itself adjusted mammal γ-aminobutyric acid
Chloride channel affinity it is relatively low, therefore, it is considered that higher to mammalian safety.However as going deep into for research,
The negative effect of ethiprole gradually shows, and ethiprole is metabolised to many metabolites in body and includes sulfone compound, sulfoxide
Object, sulfide are closed, takes off sulfinyl compound, actually these main metabolites of ethiprole are to mammal gamma-amino
The chloride channel affinity that butyric acid is adjusted is stronger, and generates ill-effect, existing experiment results proved, metabolin sulfone compound
To the toxicity higher of the toxicity of birds, vertebrate animal than ethiprole in itself, de- sulfinyl compound also has similarly
Performance.These metabolites are distributed widely in each tissue of animal by absorbing, being metabolized, draining, hydrolyzing and photolysis
In, it mainly acts on containing organ high in fat, particularly adipose tissue, adrenal gland and liver and its hetero-organization such as thyroid gland, kidney
It is dirty, animal reproduction performance, and persistent are damaged, and pass through enrichment, deposition in the tissue, passes through food chain harmful to human
Health causes people to vomit, anxious, the visible symptom such as epilepsy.Its influence to environment, the ecosystem and human health is cured
To be cured the common concern by people.The use scope that EU Committee still limited ethiprole from 2013.The United Nations's grain
Maximum residue limit has been done to ethiprole content in grain and oil, veterinary antibiotics and animal product by agriculture tissue and the World Health Organization,
Middle milk, ox kidney and birds, beasts and eggs are 0.02mg/kg, beef liver 0.1mg/kg, beef 0.5mg/kg, poultry 0.01mg/kg.China
GB2763 also provides grain and oil, veterinary antibiotics, sugar material and edible mushroom residue limits, in addition to (fresh food) corn is 0.1mg/
Outside kg, other are all 0.02mg/kg.
The remaining method of detection ethiprole mainly uses physico-chemical analysis method, mainly chromatography both at home and abroad at present, including
Liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography (GC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS),
Gas chromatography-mass spectrum method (GC-MS) etc., these methods detection ethiprole need to the pre-treatment purification process through a series of complex, it is numerous
It is trivial time-consuming, from sample pretreatment to showing that testing result at least needs 2 day time, while need large-scale special instrument and equipment and
Professional technician operates, it is impossible to which the quick detection demand of meet demand, timeliness are poor.Ethiprole immune reagent kit survey method although
Sensitivity is higher, and analytic process is relatively easy, it also requires the detection process of 2-3 hour, and microplate reader is needed to assist, no
It can carry out real time monitoring.The main means of current medicament residue rapid screening, the screening to ethiprole during ELISA test strip
There is unique advantage, be the detection technique for needing to first develop, but ethiprole test paper rapid detection method still belongs to empty at home at present
In vain.
Invention content
The technical problems to be solved by the invention are to provide a kind of high specificity, the quick of high sensitivity detects Trace Fluoride worm
Colloid gold label test strip of nitrile and preparation method thereof.
To achieve these goals, the technical solution adopted in the present invention is：
A kind of colloid gold label test strip for quickly detecting micro ethiprole, bottom are supporting layer, and middle layer is adsorption layer, are protected
Sheath is fixed on adsorption layer, adsorption layer from test lead be followed successively by adsorbing fiber layer, gold labeling antibody fibrous layer, cellulose film layer and
The absorbent material layer of handle end, wherein the carrier protein that coupling ethiprole is coated on cellulose membrane is also set as detection line
The nature controlling line of useful goat-anti or rabbit anti-mouse IgG, goat anti-rabbit igg antibody or SPA printings.
Labelled antibody in the gold labeling antibody fibrous layer is the anti-ethiprole monoclonal antibody of colloid gold label or more grams
Grand antibody.
Covered with protective film on adsorbing fiber layer, gold labeling antibody fibrous layer and absorbent material layer, adsorbing fiber layer with
It is biased to be printed with sample label at adsorbing fiber layer side 0.3-0.7cm on the corresponding protective film of gold labeling antibody fibrous layer intersection
Line.
The preparation method of the carrier protein of the coupling ethiprole is that carrying out structure to ethiprole first with Hydrolyze method changes
It makes：Under basic conditions, it is carboxyl by the cyan-hydrolysis in ethiprole, obtains CNT-COOH；Again by CNT-COOH and BSA or
OVA is coupled, and obtains the solution of coupling ethiprole carrier protein.
The preparation method of the carrier protein of the coupling ethiprole is specially：
(1) 20mg ethiprole mark product are dissolved in 100 μ L acetone, are added dropwise to the acetone soln of ethiprole under stirring
In the NaOH solution of 3mL 0.1M, for 24 hours, reaction solution gradually becomes light yellow clear solution to 60 DEG C of water-baths by muddiness at this time,
It is cooled to room temperature, then under condition of ice bath, it is 5 to adjust reacting liquid pH value with 0.1M hydrochloric acid, there is light yellow solid precipitation at this time,
Decompression filters, single to steam water washing, and drying obtains product, is named as CNT-COOH；
(2) 5mg CNT-COOH are weighed, is dissolved in 500 μ L dimethylformamides, 2.5mg 1- (3- is sequentially added under stirring
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides and 1.5mg n-hydroxysuccinimides, 0.5h is reacted at room temperature, is made
CNT-COOH is activated；
(3) it weighs 4.5mg BSA and 5mg OVA to be dissolved in respectively in 1mL phosphate buffers, after 250 μ L are activated
CNT-COOH solution is added dropwise respectively in the solution of BSA and OVA, stirs lower room temperature reaction 4h；With PBS dialysis 3d, centrifuge, receive
Collect supernatant, abandon precipitation to get to coupling ethiprole carrier protein solution, be stored in after packing -20 DEG C it is spare.
Repeatedly change dialyzate during described PBS dialysis, 6-8 times/day.
The rotating speed of the centrifugation is 5000r/min, time 5min.
A kind of preparation method of test strips, includes the following steps：
(1) it is coupled the preparation of ethiprole carrier protein
(2) preparation of anti-ethiprole monoclonal antibody or polyclonal antibody；
(3) preparation of ethiprole colloidal gold labeled monoclonal antibody；
(4) preparation of goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody；
(5) detection line is made in cellulose film layer with the ethiprole carrier protein couplet object of gained, with goat-anti or rabbit-anti
Mouse IgG or goat anti-rabbit igg antibody or SPA prepare nature controlling line in cellulose film layer；Ethiprole gold labeling antibody is used to prepare gold
Then labeling antibody fibrous layer installs supporting layer, adsorption layer and protective layer and is assembled into test strips successively.
The present invention testing principle be：
The principle of colloidal gold immune chromatography experiment is using trisodium citrate reduction gold chloride (HAuCl4) aggregate into gold
Grain due to the electrostatic interaction between gold particle and Brownian movement, makes its keep hydrosol state, and colloidal gold is rich in electronics and powerful
Electron donation.Under the conditions of colloidal solution pH8.2, colloidal gold, which is combined to form gold and mark with ethiprole antibody with non-covalent bond, to be resisted
Body (Ab-Au), gold labeling antibody is adsorbed on glass fibre cotton, and one end is with being fixed with (the detection of the coupled object of ethiprole-protein
Line) it is connected with nitrocellulose (NC) film of secondary antibody (or SPA) (nature controlling line), the other end is connected with adsorbing fiber layer.Then connect
It is made and is assembled according to design technology with other required water-absorption fibers, backing material, covering material etc., quick inspection is made
Test paper.Containing fipronil, gold labeling antibody will not be reacted with the coupled object of ethiprole protein and partly be intercepted and captured in detection sample,
Gold particle richness product and there are apparent intuitive red stripes, when gold labeling antibody not being completely combined to nature controlling line, equally will appear red
Vitta band；Contain ethiprole, ethiprole and the coupled object competitive binding gold labeling antibody of ethiprole-protein, inspection in detection sample
There are not or occurs very weak red stripes (attached drawing 1) in survey line.Quick detection test paper detection ethiprole residual, detection time only need
Several minutes.Positive beneficial effect of the present invention：
1. high specificity, high sensitivity.Test strips of the present invention are prepared based on competitive mode, sample and detection line
On target competitive binding gold label antibody.It is formed between gold particle and antibody molecule without covalent bond in gold labeling antibody, two
Person is combined by the Van der Waals force between the charges of different polarity, and colloid gold label influences very little to the specificity and affinity of antibody, and
With higher mark rate.Experiments have shown that this test paper has stronger specific and higher sensitivity, detection is limited to 10ng/
ML, with other pesticides or veterinary drug no cross reaction.Have in terms of ensuring food safety, protecting consumer health extremely important
Meaning.
It is 2. easy, quick.It, can execute-in-place without other any reagents and instrument using test strips of the present invention.By 150 μ
L sample solution is added drop-wise on test strips adsorbing fiber layer or will be inserted into testing sample solution below the test lead warning line of test strips
In, it can determine that testing result within 5 minutes or so.
3. result display is vivid, intuitive, accurate.Test strips with show brownish red " ︱ " (or " ten ", " ┬ ", " ┴ ", " ├ ",
" ┤ ") as the testing result positive and negative marker, i.e., one brownish red " ︱ " trace of display on cellulose membrane represents trace
Containing a certain concentration (test paper range estimation sensitivity concentration) and above ethiprole in sample, two brownish red " ‖ " traces, table are shown
Show that containing fipronil or content are not less than a certain concentration in test sample.Result judgement is vivid, intuitive, accurate, simple and clear, is not easy
There is false positive and false negative etc. artificially to judge by accident.
4. it saves money.Using Rapid detection test strip, without separately matching instrument and equipment and other reagents, can whenever and wherever possible into
Row detection, it is low-cost, a large amount of expensive instruments and cost of equipment can be saved.
5. it is applied widely, it is easy to utilize.It is easy to operate, the needs of different levels personnel can be met, including profession
Chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products processing, raiser and consumer individual etc., test strips tool
There are vast market prospect and apparent economic benefit and social benefit.
Description of the drawings
The specific embodiment of the present invention is described in further detail below in conjunction with attached drawing.
Fig. 1 is ELISA test strip structure diagram of the present invention.
Fig. 2 is test strips diagrammatic cross-section of the present invention.In figure, 1 is is covered in adsorbing fiber layer 2 and gold labeling antibody fibrous layer
Protective layer on 4,2 be adsorbing fiber layer, and 3 be supporting layer, and 4 be gold labeling antibody fibrous layer, and 5 be cellulose film layer, and 6 be detection
Line, 7 be nature controlling line, and 8 be the protective layer being covered on absorbent material layer 9, and 9 be absorbent material layer.
Fig. 3 is test strips schematic top plan view of the present invention.
Specific embodiment
The specific embodiment of the present invention is described in further detail with reference to embodiments.
Embodiment 1
If Fig. 1-3 is provided, the present invention quickly detects the colloid gold label test strip of micro ethiprole, and bottom is supporting layer, in
Interbed is adsorption layer, and protective layer is fixed on adsorption layer, and adsorption layer is followed successively by adsorbing fiber layer, gold labeling antibody fiber from test lead
The absorbent material layer of layer, cellulose film layer and handle end, wherein being coated with the carrier protein of coupling ethiprole on cellulose membrane
As detection line, it is additionally provided with the nature controlling line with goat-anti or rabbit anti-mouse IgG, goat anti-rabbit igg antibody or SPA printings.
Labelled antibody in the gold labeling antibody fibrous layer is the anti-ethiprole monoclonal antibody of colloid gold label or more grams
Grand antibody.
The adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made；Water-absorbent material
Layer is made of absorbent filter, and supporting layer is made of the toughness material not absorbing water；The gold of gold labeling antibody fibrous layer absorption ethiprole
Labeling antibody glass fibre cotton is made.
The cellulose film layer nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made.
The carrier protein of the coupling ethiprole is bovine serum albumin(BSA), oralbumin or keyhole limpet hemocyanin.
The detection line and nature controlling line for " ∥ " linear trace arranged in parallel or are that " 10 " font arranges trace,
Or it is " ┬ ┬ " font arrangement trace or be " ┴ ┴ " font arrangement trace or be that " ├ ├ " font arranges trace or be " ┤
┤ " fonts arrange trace.
Covered with protective film on adsorbing fiber layer, gold labeling antibody fibrous layer and absorbent material layer, adsorbing fiber layer with
It is biased to be printed with sample label at adsorbing fiber layer side 0.3-0.7cm on the corresponding protective film of gold labeling antibody fibrous layer intersection
Line.
Embodiment 2
It is made and quickly detects micro ethiprole colloid gold label test strip, prepare coupling ethiprole carrier protein and fluorine first
Worm nitrile gold labeling antibody, so as to prepare detection line and gold labeling antibody fibrous layer；Next prepares goat-anti or rabbit anti-mouse IgG (or goat-anti
Rabbit igg) antibody, it is used to prepare nature controlling line.
The present invention quickly detects the preparation method of the colloid gold label test strip of micro ethiprole, includes the following steps：
1st, the preparation of the carrier protein (i.e. ethiprole carrier protein couplet object) of coupling ethiprole
(1) Hydrolyze method carries out structure of modification to ethiprole, under basic conditions, by the cyano (- CN) in ethiprole (CNT)
It is hydrolyzed to carboxyl (- COOH) and obtains CNT-COOH.20mg ethiprole mark product are dissolved in 100 μ L acetone, by ethiprole under stirring
Acetone soln be added dropwise in the NaOH solution of 3mL 0.1M, 60 DEG C of water-baths for 24 hours, at this time reaction solution by it is muddy gradually
Become light yellow clear solution, be cooled to room temperature, then under condition of ice bath, it is 5 to adjust reacting liquid pH value with 0.1M hydrochloric acid, this
When have a light yellow solid precipitation, decompression filters, single to steam water washing, dry, obtains product, is named as CNT-COOH；
(2) 5mg CNT-COOH are weighed, is dissolved in 500 μ L dimethylformamides, 2.5mg 1- (3- is sequentially added under stirring
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides (EDC) and 1.5mg n-hydroxysuccinimides (NHS), room temperature reaction
0.5h activates CNT-COOH；
(3) 4.5mg bovine serum albumin(BSA)s (BSA) are weighed and 5mg ovalbumins (OVA) are dissolved in 1mL phosphate-buffereds respectively
In liquid, the CNT-COOH solution after 250 μ L steps (2) are activated is added dropwise respectively in the solution of BSA and OVA, stirs lower room
Temperature reaction 4h；Supernatant is collected, is abandoned with PBS dialysis 3d (period repeatedly change dialyzate, 6-8 times/day), 5000r/min centrifugation 5min
Precipitate to get to coupling ethiprole carrier protein solution, be stored in after packing -20 DEG C it is spare.
2nd, the preparation of anti-ethiprole monoclonal antibody or polyclonal antibody
It is prepared by monoclonal antibody：Exempted from the ethiprole carrier protein couplet object of preparation with the dosage of 20 μ g albumen every time/200 μ L/
6~8 week old Balb/C mouse of epidemic disease, 4~6 points of injections of dorsal sc point, exempt from 4 times, 4 weeks immunization interval time altogether.Head exempts from, and use is sterile
Ethiprole carrier protein couplet object prepared by PBS dilutions, mixes with equivalent Freund's complete adjuvant (FCA), fully emulsified；Reinforcement is exempted from
Epidemic disease dilutes ethiprole carrier protein couplet object with sterile PBS, is mixed with equivalent incomplete Freund's adjuvant (FIA), fully emulsified,
The 4th measures serum antibody titer and inhibits potency after being immunized 10 days, screening potency is high, and the good mouse of inhibition is as fusion
Mouse.It is superpower immune to fusion mouse progress, 100 μ g ethiprole carrier protein couplet objects are diluted to 100 μ g with sterile PBS
The μ L of albumen/200, are not added with adjuvant and are directly injected intraperitoneally.Sinus under immune mouse socket of the eye is taken a blood sample after 3~4 days, detaches positive serum；
De- neck is lethal, impregnates 5~10min of mouse disinfection body surfaces with the alcohol of 75% (v/v), sterile to take its spleen, and spleen is shredded simultaneously
Grinding is filtered through 120 mesh nylon gauzes, and 1000r/min centrifugation 10min collect splenocyte.By the splenocyte of separation and NS0 bones
Myeloma cells press 10:1 ratio mixing, 1000r/min centrifugations 10min abandon supernatant, and cell precipitate is delayed in 37 DEG C of water-baths
It is added in the slow 50%PEG4000 (v/v) for adding in 0.7~1.0mL, 1min, preceding 30s adds 0.1~0.3mL, intermediate 15s to add 0.2
~0.4mL, last 15s are added；Then 1640 culture medium 15mL of serum-free is slowly added into, to terminate the effect of PEG, 37 DEG C of water-baths
5~10min, 1000r/min centrifugation 10min abandon supernatant, cell precipitate are resuspended in HAT Selective agar mediums, and add in 96
In porocyte culture plates (8~10 pieces), the μ L/ holes of 100 μ L~200 are placed in 37 DEG C, 5% CO2It is cultivated in incubator.Culture 10
It~14 days, carries out positive hole sizer with indirect elisa method and selects, the hole that strong positive, inhibiting rate are high, cell growth is vigorous is selected to carry out 2
Secondary limited dilution cloning then expands culture, establishes hybridoma cell strain.
Ascites method batch is induced in vivo and prepares antibody, takes SPF grades of BALB/C mices, 400 μ L/ of intraperitoneal injection atoleine
Only, after 7~10 days, being diluted to a concentration of 1 × 10 with culture medium RMPI-16406The hybridoma of a cell/mL, by 500
μ L/ are only injected into abdominal cavity.It treats mouse peritoneal enlargement, when skin is tight, takes ascites, 3000r/min centrifugation 5min, it is heavy to abandon
It forms sediment.Then sad ammonium sulfate method purifies ascites, and adding in 4mL ethyl sodium buffer solutions (pH 4.0) in 1mL ascites dilutes abdomen
The ethyl sodium pH value of buffer solution of ascites is adjusted to 4.5 by water with the NaOH solution of 1M.Then it is slowly stirred and adds in 130 μ L
Octanoic acid is stirred to react 30min at room temperature, and 6000r/min centrifugation 30min go to precipitate, and supernatant is filtered to remove impurity with filter paper, filter
Liquid and the phosphate buffers (PBS) of 10 times of concentrations by volume 9:1 mixing, i.e. filtrate are under 1 times of PBS ionic environment, so
The NaOH tune pH to 7.4 of 1M is used afterwards, is put into ice bath and is cooled down.Ammonium sulfate powder is added in by the concentration of 0.2778g/mL, is stirred at 4 DEG C
Mix reaction 2h, 6000r/min centrifugation 20min, remove supernatant, 1mL PBS dissolving precipitations, PBS flowings dialysis 3 days, 6000r/min from
Heart 10min abandons precipitation, -20 DEG C freeze it is spare.After testing, prepared monoclonal antibody can specifically be reacted with ethiprole, parent
Reach 10 with force constant10-1012L/mol, available for preparing gold labeling antibody fibrous layer.
Mostly anti-preparation：New Zealand White Rabbit is immunized with ethiprole carrier protein couplet object, immunizing dose is 200 μ g eggs every time
In vain/1mL/ only, exempt from 4 times, 3~5 weeks immunization interval time altogether by 4~6 points of injections of dorsal sc point.Head exempts from, and is diluted with sterile PBS
The ethiprole carrier protein couplet object of preparation, mixes with equivalent Freund's complete adjuvant (FCA), fully emulsified；Booster immunization, with nothing
Bacterium PBS dilutes ethiprole carrier protein couplet object, is mixed with equivalent incomplete Freund's adjuvant (FIA), fully emulsified.For the last time
10~15 days after immune, it is surveyed with ELISA method determine potency and reach 105More than when, take a blood sample and separate and collect hyper-immune serum.Saturation sulphur
Acid ammonium salt analysis method extraction IgG antibody, takes 1 portion of hyper-immune serum to add 2 parts of PBS mixings, adds isometric saturated ammonium sulfate solution mixing, put
4 DEG C of refrigerator 12h, 4 DEG C, 2500r/min centrifugation 15min are abandoned supernatant, then dissolved and precipitated with appropriate PBS, add saturated ammonium sulfate solution
To final concentration 33%, 4 DEG C of refrigerator 2h are put, 4 DEG C, 2500r/min centrifugation 15min are abandoned supernatant, dissolved and precipitated with appropriate PBS, put 4
PBS dialyses 3 days in DEG C dialysis cabinet, and liquid is changed for several times in centre, and 4 DEG C, 12000r/min centrifugation 15min collect supernatant, obtain the anti-of purifying
Ethiprole polyclonal antibody, -20 DEG C freeze, and are used to prepare gold labeling antibody fibrous layer.
3rd, the preparation of ethiprole gold labeling antibody and gold labeling antibody glass fibre cotton
Colloidal gold solution is prepared using reduction of sodium citrate method.99mL ultra-pure waters are added to clean 200mL with a scale
In conical flask, conical flask is placed on heating magnetic stirring apparatus and heats and stir, and adds in 1mL 1% (w/v) chlorauric acid solution, heating
To boiling, the citric acid three sodium solution of 1.6mL 1% (w/v) is then rapidly added, continues to be heated with stirring to solution colour by shallow
Yellow becomes peony, continues to boil 3-5min (paying attention to adding ultra-pure water holding liquor capacity during boiling), is cooled to room temperature, uses
The K of 0.1mol/L2CO3PH value is adjusted to 8.2 or so, colloidal gold solution is made, room temperature is kept in dark place spare.By ethiprole antibody
1mg/mL is diluted to PBS, the ratio of 1mL colloidal gold solutions is added in 20 μ L1mg/mL antibody-solutions, a concentration of 1mg/mL's
Antibody-solutions are added dropwise in colloidal gold solution, react at room temperature 10min, add in the PEG10000 of 20% (v/v) to final concentration
It is 0.05%, 4 DEG C, 12000r/min centrifugation 1h abandon supernatant, obtain the gold labeling antibody protein mixture of preliminary purification, precipitation use etc.
Volume 20mmol/L borate buffers (BSA for containing 1% (w/v)) are resuspended, and 4 DEG C, 12000r/min centrifugation 1h abandon supernatant, precipitate
With TB solution (Na2B4O7·10H2O0.7627g, BSA1g, sucrose 3g, NaN30.03g, 80mL DDW are fully dissolved and are settled to
100mL, then with 0.2 μm of membrane filtration, 4 DEG C of preservations) to be resuspended be 10 × concentrate (before volume be centrifugation for the last time after resuspension
1/10, i.e. 10 times of concentrates), 4 DEG C save backup.During metal spraying, 10 × gold labeling antibody concentrate TB is diluted 5 times, becomes 2
2 × gold labeling antibody concentrate is sprayed on glass fibre cotton by × gold labeling antibody concentrate with spraying instrument by 5 μ L/cm, and 4 DEG C low
Temperature vacuum drying, prepares ethiprole gold labeling antibody fibrous layer.
4th, the preparation of goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody
Rabbit or sheep is immunized in the ethiprole negative mice serum IgG purified with saturated ammonium sulfate, and (or feminine gender rabbit anteserum IgG is immunized
Sheep), ethiprole rabbit polyvalent antibody is the same with preparing for method, for the system of Quality Control trace in micro ethiprole Rapid detection test strip
It is standby.Quality Control trace can also be spraying SPA (staphylococcal protein A).
5th, the assembling of test strips
Detection line is made in cellulose film layer with the ethiprole carrier protein couplet object of gained, with goat-anti or rabbit anti-mouse
IgG or goat anti-rabbit igg antibody or SPA prepare nature controlling line in cellulose film layer；By adsorbing fiber layer, ethiprole colloidal gold mark
Note antibody fibrous layer, cellulose film layer and layers of absorbent material are assembled on supporting layer, then assemble protective layer successively, and test paper is made
Item.
Detect the detection reaction principle of the colloid gold label test strip of micro ethiprole：
By 150 μ L sample solution be added drop-wise on test strips adsorbing fiber layer or by the test lead warning line (MAX) of test paper with
After lower insertion testing sample solution, solution to be measured is driven by siphonage in ethiprole to be measured and gold labeling antibody glass fibre cotton
Gold labeling antibody spread together to cellulose film layer, and eventually penetrate the absorbent material layer of handle end.It is to be measured in diffusion process
Ethiprole can be combined with the gold labeling antibody in gold labeling antibody fibrous layer, and then close the antigen binding of ethiprole in gold labeling antibody
Point prevents gold labeling antibody and the detection trace (detection line) of coupling ethiprole carrier protein on cellulose membrane from combining, in test strips
It can not show detection trace, and sheep or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody can then be combined with gold labeling antibody, form palm fibre
Red Quality Control trace band (nature controlling line) " ︱ ", i.e. brownish red band " ︱ " trace represent to be positive.If otherwise nothing in sample solution
Ethiprole cannot then prevent gold labeling antibody from being combined with being coupled the detection trace of ethiprole carrier protein on cellulose membrane, test strips
Show brownish red detection trace band " ︱ "；Similary goat-anti or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody also can be with gold labeling antibodies
With reference to display brownish red Quality Control trace band " ︱ " forms two brownish red bands " ‖ " and represented to be negative.If nature controlling line is not reddish brown
Colour band is shown, then shows that test strips have failed.
Experimental example, sensitivity and specificity detection
With a concentration of 1ng/mL, 2ng/mL, 4ng/mL, 6ng/mL, 8ng/mL, 10ng/mL, 20ng/mL and 40ng/mL
Ethiprole solution is sample, and ethiprole detection is carried out using ethiprole colloid gold label test strip.After five minutes, the results show that
All ELISA test strip lines do not develop the color when 10ng/mL, 20ng/mL and 40ng/mL, and there is 90% test strips inspection in when 8ng/mL
Survey line does not develop the color, and the ELISA test strip line that when 6ng/mL has 50% does not develop the color, the test strips inspection of 1ng/mL, 2ng/mL and 4ng/mL
Survey line develops the color, therefore, it is determined that the range estimation minimal detectable concentration of ethiprole colloid gold label test strip is 10ng/mL, sensitivity setting
For 10ng/mL.
With the Bravo of a concentration of 20 μ g/mL, glyphosate, phosmet, chlopyrifos, parathion, malathion and enemy enemy
Fear for sample, be detected using ethiprole colloid gold label test strip.The results show that the detection line of each test strip is aobvious
Color is not suppressed, and illustrates the test strips high specificity of the present invention, with other pesticide no cross reactions.
Claims (8)
1. a kind of colloid gold label test strip for quickly detecting micro ethiprole, bottom are supporting layer, middle layer is adsorption layer, protection
Layer is fixed on adsorption layer, it is characterized in that：Adsorption layer is followed successively by adsorbing fiber layer, gold labeling antibody fibrous layer, fiber from test lead
The absorbent material layer of plain film layer and handle end, wherein being coated with the carrier protein of coupling ethiprole on cellulose membrane as detection
Line is additionally provided with the nature controlling line with goat-anti or rabbit anti-mouse IgG, goat anti-rabbit igg antibody or SPA printings.
2. test strips according to claim 1, it is characterized in that：Labelled antibody in the gold labeling antibody fibrous layer is colloid
The anti-ethiprole monoclonal antibody or polyclonal antibody of gold label.
3. test strips according to claim 1, it is characterized in that：In adsorbing fiber layer, gold labeling antibody fibrous layer and water-absorption material
Covered with protective film on the bed of material, it is fine that absorption is biased on adsorbing fiber layer protective film corresponding with gold labeling antibody fibrous layer intersection
Sample mark line is printed at dimension layer side 0.3-0.7cm.
4. according to claim 1-3 any one of them test strips, it is characterized in that：The carrier protein of the coupling ethiprole
Preparation method is to carry out structure of modification to ethiprole first with Hydrolyze method：Under basic conditions, by the cyan-hydrolysis in ethiprole
For carboxyl, CNT-COOH is obtained；CNT-COOH and BSA or OVA is coupled again, obtains the solution of coupling ethiprole carrier protein.
5. test strips according to claim 4, it is characterized in that：The preparation method of the carrier protein of the coupling ethiprole
Specially：
(1) 20mg ethiprole mark product are dissolved in 100 μ L acetone, the acetone soln of ethiprole is added dropwise to 3mL under stirring
In the NaOH solution of 0.1M, for 24 hours, reaction solution gradually becomes light yellow clear solution to 60 DEG C of water-baths by muddiness at this time, cooling
To room temperature, then under condition of ice bath, it is 5 to adjust reacting liquid pH value with 0.1M hydrochloric acid, there is light yellow solid precipitation at this time, is depressurized
It filters, single to steam water washing, drying obtains product, is named as CNT-COOH；
(2) 5mg CNT-COOH are weighed, is dissolved in 500 μ L dimethylformamides, 2.5mg 1- (3- diformazans is sequentially added under stirring
Aminopropyl) -3- ethyl-carbodiimide hydrochlorides and 1.5mg n-hydroxysuccinimides, 0.5h is reacted at room temperature, makes CNT-
COOH is activated；
(3) it weighs 4.5mg BSA and 5mg OVA to be dissolved in respectively in 1mL phosphate buffers, the CNT- after 250 μ L are activated
COOH solution is added dropwise respectively in the solution of BSA and OVA, stirs lower room temperature reaction 4h；With PBS dialysis 3d, centrifuge, in collection
Clearly, abandon precipitation to get to coupling ethiprole carrier protein solution, be stored in after packing -20 DEG C it is spare.
6. test strips according to claim 5, it is characterized in that：Repeatedly change dialyzate during described PBS dialysis, 6-8 times/
My god.
7. test strips according to claim 5, it is characterized in that：The rotating speed of the centrifugation is 5000r/min, and the time is
5min。
8. a kind of preparation method of test strips as claimed in claim 4, which is characterized in that include the following steps：
(1) it is coupled the preparation of ethiprole carrier protein
(2) preparation of anti-ethiprole monoclonal antibody or polyclonal antibody；
(3) preparation of ethiprole colloidal gold labeled monoclonal antibody；
(4) preparation of goat-anti or rabbit anti-mouse IgG or goat anti-rabbit igg antibody；
(5) detection line is made in cellulose film layer with the ethiprole carrier protein couplet object of gained, with goat-anti or rabbit anti-mouse
IgG or goat anti-rabbit igg antibody or SPA prepare nature controlling line in cellulose film layer；It is anti-that ethiprole gold labeling antibody is used to prepare gold mark
Then body fibrous layer installs supporting layer, adsorption layer and protective layer and is assembled into test strips successively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810005478.1A CN108226489A (en) | 2018-01-03 | 2018-01-03 | A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810005478.1A CN108226489A (en) | 2018-01-03 | 2018-01-03 | A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108226489A true CN108226489A (en) | 2018-06-29 |
Family
ID=62642865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810005478.1A Pending CN108226489A (en) | 2018-01-03 | 2018-01-03 | A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108226489A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111187346A (en) * | 2020-01-15 | 2020-05-22 | 江苏大学 | Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof |
| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414390A (en) * | 2002-12-13 | 2003-04-30 | 江苏省农业科学院植物保护研究所 | Fipronil immune detecting method |
| CN101100486A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial antigen, antibody and use thereof |
| CN101100457A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil hapten compound, synthetic method and use thereof |
| CN101100456A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use |
| CN103454420A (en) * | 2013-08-03 | 2013-12-18 | 河南百奥生物工程有限公司 | Test strip for rapidly detecting trace sulfachlorpyridazine and preparation method thereof |
-
2018
- 2018-01-03 CN CN201810005478.1A patent/CN108226489A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414390A (en) * | 2002-12-13 | 2003-04-30 | 江苏省农业科学院植物保护研究所 | Fipronil immune detecting method |
| CN101100486A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial antigen, antibody and use thereof |
| CN101100457A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil hapten compound, synthetic method and use thereof |
| CN101100456A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use |
| CN103454420A (en) * | 2013-08-03 | 2013-12-18 | 河南百奥生物工程有限公司 | Test strip for rapidly detecting trace sulfachlorpyridazine and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 董健等: "，氟虫腈半抗原设计及抗体制备", 《江苏农业学报》 * |
| 郑尊涛: "杀虫剂吡虫啉和氟虫腈的酶联免疫吸附测定（ELISA）技术研究", 《中国优秀硕士论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111187346A (en) * | 2020-01-15 | 2020-05-22 | 江苏大学 | Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof |
| CN111187346B (en) * | 2020-01-15 | 2023-05-05 | 江苏大学 | Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof |
| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
| CN113063938B (en) * | 2021-03-13 | 2023-09-12 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107941785A (en) | A kind of surface-enhanced Raman gold label test strip for quickly detecting micro ethiprole and preparation method thereof | |
| CN108226490A (en) | A kind of quantum dot test strips for quickly detecting micro ethiprole and preparation method thereof | |
| CN102798719B (en) | Test paper strip for rapidly detecting traces of chlorothalonil and preparation method thereof | |
| CN104251904B (en) | A kind of test paper of Quantitative detection pyrethrin pesticide | |
| CN102654500A (en) | Detecting reagent kit used for detecting quinoxalinone-2-carboxylic acid and method | |
| CN105652004B (en) | A kind of TMP haptens and its collaurum detection means and preparation method thereof | |
| CN105044365B (en) | The preparation method of the time resolved fluoro-immunoassay test paper of detection enrofloxacin residual | |
| CN108226489A (en) | A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof | |
| CN103439514A (en) | Pesticide and veterinary drug multi-residue detection method based on microarray detection chip | |
| CN102798720A (en) | Immune chromatography test paper for quantitative determination of clenbuterol based on up-conversion fluorescent nanoparticle label and preparation method thereof | |
| CN102818895A (en) | Test strip for rapidly detecting lincomycin residues and preparation method of test strip | |
| CN102495212B (en) | Method and test paper for rapidly detecting microcystins | |
| CN101429241A (en) | Penicillin and carrier protein couplet product, method for producing beta-lactam penicillin antibody, and uses thereof | |
| CN102455357B (en) | Kit and method for detecting beta-lactamase | |
| CN102297970A (en) | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof | |
| CN101408545A (en) | Melamine fast detecting test paper bar and test paper card | |
| CN104101707B (en) | 2,4 dichlorophenoxyacetic acid residual Rapid detection test strip | |
| CN102020713A (en) | Immune colloidal gold test strip used for detecting aureomycin residue and preparation method thereof | |
| CN106831498A (en) | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN201051101Y (en) | Quick testing paper for ractopamine residue | |
| CN102507930A (en) | Kit for quickly detecting trace imidacloprid residue | |
| CN101979511B (en) | Heavy metal zinc-resistant monoclonal antibody | |
| CN1811448A (en) | Phenobarbital fast detecting test paper bar | |
| CN103454420B (en) | Test strip for rapidly detecting trace sulfachlorpyridazine and preparation method thereof | |
| CN103353524A (en) | Test strip for rapidly detecting microscale olaquindox |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180629 |