CN109061155A - A kind of test strips and its preparation method and application detecting metalaxyl - Google Patents
A kind of test strips and its preparation method and application detecting metalaxyl Download PDFInfo
- Publication number
- CN109061155A CN109061155A CN201811104762.0A CN201811104762A CN109061155A CN 109061155 A CN109061155 A CN 109061155A CN 201811104762 A CN201811104762 A CN 201811104762A CN 109061155 A CN109061155 A CN 109061155A
- Authority
- CN
- China
- Prior art keywords
- metalaxyl
- test strips
- sample
- coated
- pad
- 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.)
- Granted
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
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/577—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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (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)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of test strips and its preparation method and application for detecting metalaxyl, which includes sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；It is coated with the detection line of metalaxyl hapten-carrier protein conjugate it is characterized by: having on the reaction film and is coated with the nature controlling line of sheep anti mouse antiantibody, metalaxyl monoclonal antibody-colloid gold label object is coated in the conjugate release pad.The metalaxyl haptens is to generate nitre phenyl metalaxyl by N- (2,6- 3,5-dimethylphenyl) methyl lactamine and (4-nitrophenoxy) excess acetyl chloride, then obtain by zinc powder reduction.The present invention also provides a kind of methods using metalaxyl in above-mentioned test strips test sample.Test strips and detection method provided by the invention have the advantages that high sensitivity, specificity are good, easy to operate, detect fast, at low cost, the not examined equipment limit of speed, are able to achieve the quick detection and on-site supervision of metalaxyl in batch samples.
Description
Technical field
The present invention relates to the detection of metalaxyl, test strips and preparation method thereof of specifically a kind of detection metalaxyl and answer
With it is especially suitable for the detections of metalaxyl residue in tobacco leaf.
Background technique
Metalaxyl (Metalaxyl) is a kind of strong absorbability substituted benzene acid amide fungicides, chemical name N- (2- methoxyl group second
Acyl group)-N- (3,5-dimethylphenyl)-racemization-aminobenzoic acid formic acid, belong to acid amide fungicides, high-efficiency low-toxicity is mainly used for oomycetes
Guiding principle, Phycomycetes, fungus-caused various downy mildew, late blight, early blight, samping off and withered rotten disease, fruit rot etc..Main suppression
The synthesis of germ mycelia vivo protein processed, makes its nutritional deficiency, is unable to normal growth and dead.Its interior suction and penetration are very
By force, after application 30min can in plant up and down Bidirectional Conduction, have protection and therapeutic effect to disease plant, to prevention and treatment melon
Fruits and vegetables, white viral disease, the epidemic disease of tobacco have good therapeutic effect.
But metalaxyl is one of important pollutant in environment, and can the mankind be caused with potential health threat, and pollution ecology
Environment, so its residue problem in water fruits and vegetables, tobacco leaf production receives more and more attention.China is directed to different works
Object has formulated metalaxyl maximum residue limit standard, wherein cucumber, capsicum, tomato maximum residue limit be 0.5 mg/kg,
The maximum residue limit of brown rice is 0.1 mg/kg, other cereal maximum residue limit are 0.05 mg/kg.International tobacco science is ground
The guiding residue limits for studying carefully metalaxyl in Cooperation Centre (CORESTA) regulation tobacco are 2 mg/kg, in actual production, with 2
Mg/kg is as tobacco maximum residue limit criterion.
Currently, mainly having gas chromatography-mass spectrometry, high-efficient liquid phase color to the detection method of metalaxyl residue both at home and abroad
Spectrum-Mass Spectrometry, gas chromatography, high performance liquid chromatography.It is excellent that instrumental method has detection sensitivity height, high specificity etc.
Gesture, but detection Sample pretreatment is cumbersome, time-consuming, sample also needs extraction and purified treatment, while instrument detection method needs to hold high
Expensive large-scale instrument and equipment is equipped with the detection technique personnel of profession, is operated and managed, and can not carry out live extensive inspection
It surveys, poor in timeliness, it is difficult to promote.Therefore, develop a kind of not examined equipment limit and can be realized to batch samples into
The product and method that row quickly detects become problem in the urgent need to address.
Patent of invention (201510530296.2) discloses test paper and its application of detection metalaxyl residue, and the present invention is therewith
Maximum difference is that the structure of haptens and preparation method are significantly different.It is well known that establishing the immune of small molecule compound
The key of analysis method is can to prepare to have high-affinity and highly selective antibody to small molecule compound, and synthesize end
Base is functional group and the haptens of the linking arm with certain length is to prepare anti-small molecule compound antibody, establish accordingly
The significant process of immunoassay method.Difference on haptens and any structure of analyte substance of interest, such as molecular size, shape
Topological sex character including shape, ingredient, configuration, conformation, polarity, cloud density etc., all may strong influence corresponding antibodies
Property.It can design and synthesize the better haptens of performance, effect, to obtain the good antibody of high sensitivity, specificity, just
It is present invention emphasis of interest.
Summary of the invention
It is an object of the invention to overcome existing for the method for existing detection metalaxyl to device dependence height, and not
The characteristics of can be realized the quick detection to batch samples, provide a kind of easy to operate, high sensitivity, detection speed it is fast, at
The test strips and its preparation method and application of this low, not examined equipment limit, with realize to large batch of metalaxyl sample into
Row quickly detection and on-site supervision.
In order to achieve the object of the present invention, the present invention provides a kind of test strips for detecting metalaxyl, which includes
Sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；Wherein, have on the reaction film and be coated with metalaxyl
The detection line of hapten-carrier protein conjugate and the nature controlling line for being coated with sheep anti mouse antiantibody, in the conjugate release pad
It is coated with metalaxyl monoclonal antibody-colloid gold label object, the metalaxyl monoclonal antibody is with metalaxyl hapten-carrier
Protein conjugate is prepared as immunogene, the metalaxyl hapten-carrier protein conjugate by metalaxyl haptens with
Carrier protein couplet obtains, and the carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or people
Seralbumin, the metalaxyl haptens are by N- (2,6- 3,5-dimethylphenyl) methyl lactamine and (4-nitrophenoxy) second
Acyl chloride reaction generates nitre phenyl metalaxyl, then is obtained by zinc powder reduction, molecular structural formula are as follows:
。
The metalaxyl haptens specific the preparation method is as follows:
1) 2.00 g of N- (2,6- 3,5-dimethylphenyl) methyl lactamine is taken, adds pyridine sufficiently to dissolve, adds 2.30 g (4- nitrobenzene
Oxygroup) chloroacetic chloride, stirring, oil bath heating, 60 DEG C of reaction 2 h, TLC detections, raw material fundamental reaction is complete, stops reaction, restores
To room temperature, revolving removes pyridine, adds 60 mL water, with 1 mol/L salt acid for adjusting pH value to 5,80 mL ethyl acetate is added to extract,
Organic phase anhydrous sodium sulfate is dry, upper silicagel column, using the ethyl acetate that volume ratio is 1:10/n-hexane elution separation, obtains
3.68 g of intermediate product nitre phenyl metalaxyl；
2) 3.60 g of intermediate product nitre phenyl metalaxyl is taken, adds ethyl alcohol to dissolve, obtains A liquid；1.80 g of zinc powder is taken, 10 mL is added to steam
Distilled water adds 0.5 mL of dilute hydrochloric acid, 60 DEG C of 20 min of activation, and A liquid is added, and continues to stir 3 h, TLC is detected, and raw material total overall reaction is complete
At stopping reaction filtering, removes zinc powder, and filtrate is evaporated, and adds 50 mL water, adjusts pH value to 7 with sodium carbonate, adds 50 mL acetic acid
Ethyl ester extraction, organic phase washing, anhydrous sodium sulfate drying are evaporated, and are tied again using methylene chloride/n-hexane that volume ratio is 1:2
Crystalline substance obtains 3.22 g of metalaxyl haptens product.
The sheep anti mouse antiantibody is to carry out immune prepare to goat using source of mouse antibody as immunogene.
The sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on bottom plate, the conjugate
Release pad 1/3 ~ 1/2 is capped under sample absorption pad.
The bottom plate is the material that PVC bottom plate or other hard do not absorb water；The sample absorption pad is suction strainer paper or oil strain
Paper；The conjugate release pad is mineral wool or polyester material；The water absorption pad is blotting paper；The reaction film is cellulose nitrate
Plain film or cellulose acetate film.
A method of preparing above-mentioned test strips, comprising the following steps:
1) preparation is coated with metalaxyl monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with metalaxyl hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
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.
More specifically, the process of test strips is prepared are as follows:
1) N- (2,6- 3,5-dimethylphenyl) methyl lactamine and (4-nitrophenoxy) excess acetyl chloride are generated into nitre phenyl first frost
Spirit, then by zinc powder reduction, prepare metalaxyl haptens；
2) by metalaxyl haptens and carrier protein couplet, metalaxyl hapten-carrier protein conjugate is prepared；
3) mouse is immunized with metalaxyl hapten-carrier protein conjugate, by mouse boosting cell and myeloma cell by fusion,
Screening obtains the hybridoma cell strain of secretion metalaxyl monoclonal antibody；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) metalaxyl hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in the detection line (T) of reaction film respectively
On nature controlling line (C)；
6) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
7) the metalaxyl monoclonal antibody of preparation is added in the colloidal gold of preparation, obtains metalaxyl monoclonal antibody-colloid
Golden marker；
8) metalaxyl monoclonal antibody-colloid gold label object is sprayed in conjugate release pad, takes out, sets after 37 DEG C of 1 h of baking
It is saved backup in dry environment；
9) sample absorption pad is used and is delayed containing 0.5% bovine serum albumin(BSA) (mass fraction), the 0.1 mol/L phosphate that pH value is 7.2
Fliud flushing impregnates 2 h, dries 2 h at 37 DEG C；
10) upper sample absorption pad, conjugate release pad, reaction film, water absorption pad, conjugate release pad are pasted in order on bottom plate
There is 1/3 region to be absorbed by the sample pad covering from starting point.It is finally cut into the small item of 3 mm wide, adds plastic casing, is vacuum-packed, 4 ~ 30
It can be reserved for 12 months under the conditions of DEG C.The 1/3 of conjugate release pad be absorbed by the sample pad covering can extend testing result observation when
Between, sample absorption pad can be made to will test liquid and fully absorb and sufficiently reacted with gold labeling antibody, to reduce error.
Using the method for metalaxyl in above-mentioned test strips test sample, include the following steps:
1) pre-treatment is carried out to sample；
2) it is detected with test strips；
3) analysis detection result.
The test strips of detection metalaxyl of the invention are using the antibody antigen reaction of high degree of specificity and immunochromatographiassays assays
Metalaxyl monoclonal antibody-colloid gold label object is fixed in conjugate release pad by technology, and the metalaxyl in sample is flowing
In the process, in conjunction with metalaxyl monoclonal antibody-colloid gold label object in conjugate release pad, metalaxyl-antibody-glue is formed
Body gold marker.The metalaxyl hapten-carrier protein conjugate on metalaxyl and reaction film detection line in sample, which competes, to be tied
Close metalaxyl monoclonal antibody-colloid gold label object, judged in analyte sample fluid according to the detection line red stripes depth whether
Contain metalaxyl residue.
When detection, sample instills sample absorption pad after processing, when the concentration of metalaxyl in the sample lower than detection limit or
When being zero, monoclonal antibody-colloid gold label object meeting and the metalaxyl haptens-load being fixed on reaction film in chromatography process
Body protein conjugate combines, and respectively occurs a red stripes at detection line (T) and nature controlling line (C), and the colour developing of T line is more aobvious than C line
Color depth is consistent with the colour developing of C line；If the concentration of metalaxyl in the sample is equal to or higher than detection limit, monoclonal antibody-colloid
Golden marker can all be combined with metalaxyl, thus because competitive reaction will not be with metalaxyl hapten-carrier albumen at T line
Conjugate is combined without red stripes occur or developing the color than C line shallow.As shown in Figure 2.
It is negative: when nature controlling line (C) shows that red stripes, detection line (T) also show that red stripes, and (T) line simultaneously
When color is close or is deeper than (C) line, it is judged to feminine gender.
Positive: when nature controlling line (C) shows red stripes, and detection line (T) does not develop the color or (T) line color is shallower than (C) line
When, it is judged to the positive.
It is invalid:, should whether nature controlling line (C) does not show red stripes, then no matter detection line (T) shows red stripes
Test strips are judged in vain.
In the present invention and 201510530296.2 in the molecular structure of haptens and selected hapten molecule structure
Decorating site is significantly different, and the space structure that different decorating sites is formed by hapten transformation object is different, i.e., with carrier knot
The antigenic determinant exposed on the artificial antigen surface generated after conjunction is different, causes the antibody generated in potency, affinity
With all had differences in specificity.
Haptens of the invention has appropriate terminal reactive group, and decorating site and spacer length selection are suitable, and energy
The molecular structure of metalaxyl is utmostly simulated, the test strips developed based on this haptens have high sensitivity, specificity
Strong feature.Test strips of the invention simultaneously are at low cost, easy to operate, detection time is short, not examined equipment limit, fits
Close the advantages of various units use, storage is simple, long shelf-life.It is easy, fast with the method for test strips of the present invention detection metalaxyl
Fast, intuitive, accurate, applied widely, at low cost, easy popularization and use.
Detailed description of the invention
Fig. 1 is test strips the schematic diagram of the section structure, in figure: 1, sample absorption pad；2, conjugate release pad；3, reaction film；
4, water absorption pad；5, detection line；6, nature controlling line；7, bottom plate；
Fig. 2 is test strips testing result process decision chart；
Fig. 3 is metalaxyl hapten synthesis figure (figure is as Figure of abstract).
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.In addition, the range that those skilled in the art limits in the appended claims
Interior to carry out various changes or modification to the present invention, these are changed or modification should equally fall into the protection scope of invention.
Embodiment 1 detects the preparation of the test strips of metalaxyl
The preparation method of the test strips mainly comprises the steps that
1) preparation is coated with metalaxyl monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with metalaxyl hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
And 2) 3) 1) conjugate release pad, reaction film and the sample absorption pad, water absorption pad and the PVC bottom plate that prepare are assembled into examination
Paper slip.
Substep narration in detail below:
1, the synthesis (synthetic route is shown in Fig. 3) and identification of metalaxyl haptens
2.00 g of N- (2,6- 3,5-dimethylphenyl) methyl lactamine is taken, adds pyridine sufficiently to dissolve, adds 2.30 g (4- nitrobenzene oxygen
Base) chloroacetic chloride, stirring, oil bath heating, 60 DEG C of reaction 2 h, TLC detections, raw material fundamental reaction is complete, stops reaction, restores extremely
Room temperature, revolving remove pyridine, add 60 mL water, with 1 mol/L salt acid for adjusting pH value to 5, add 80 mL ethyl acetate to extract, have
Machine phase anhydrous sodium sulfate is dry, upper silicagel column, using the ethyl acetate that volume ratio is 1:10/n-hexane elution separation, obtains
Between 3.68 g of product nitre phenyl metalaxyl, yield 98.92%；
3.60 g of intermediate product nitre phenyl metalaxyl is taken, adds ethyl alcohol to dissolve, obtains A liquid；1.80 g of zinc powder is taken, 10 mL is added to distill
Water adds 0.5 mL of dilute hydrochloric acid, 60 DEG C of 20 min of activation, and A liquid is added, and continues to stir 3 h, TLC is detected, and raw material total overall reaction is complete
At stopping reaction filtering, removes zinc powder, and filtrate is evaporated, and adds 50 mL water, adjusts pH value to 7 with sodium carbonate, adds 50 mL acetic acid
Ethyl ester extraction, organic phase washing, anhydrous sodium sulfate drying are evaporated, and are tied again using methylene chloride/n-hexane that volume ratio is 1:2
Crystalline substance obtains 3.22 g of metalaxyl haptens product, yield 96.99%.
Nuclear-magnetism identification1H NMR(CDCl3, 300MHz) and δ: 4.778 (2H, q, J=7.047), 3.646(3H), 1.104
(3H, d, J=7.047), 4.52(t, 1H), 7.313(1H, dd, J=7.888), 2.260(t, 6H), 7.41(1H, dd,
J=7.888), 6.865(1H, t, J=7.888), 6.858(1H, ddd, J=8.804, J=0.545, J=0.000),
6.272(t 2H).In map, chemical shift δ=6.272 are the resonance absorbing peak of phenyl ring amino hydrogen on spacerarm, δ=
6.858,6.856 be phenyl ring hydrogen on spacerarm resonance absorbing peak, the presence at these peaks proves that spacerarm be coupled successfully, and first is white
Clever haptens structure is correct.
2, the synthesis and identification of metalaxyl coupled antigen
Immunogene preparation --- metalaxyl haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
It takes 8 mg metalaxyl haptens to add 0.1 mL of dilute hydrochloric acid of 1 mol/L, adds 0.8 mL of distilled water, 0 ~ 5 DEG C of low temperature stirs
20 min are mixed, 0.1 mL of aqueous solution containing 1.7 mg sodium nitrites is added, continues to stir 1 h, obtains A liquid；50 mg BSA are taken,
0.1 mol/L sodium carbonate liquor, 6 mL is added to dissolve, 0 ~ 5 DEG C of stirring 20 min of equilibrium temperature obtains B liquid, A drop is added to B liquid
In, the reaction was continued 2 h.Stopping reaction, 0.02 mol/L PBS, 3 d of dialysis change liquid three times daily, dispense, obtain immunogene ,-
20 DEG C of preservations, it is spare.
Coating antigen preparation --- metalaxyl haptens and ovalbumin (OVA) coupling obtain coating antigen.
It takes 6 mg metalaxyl haptens to add 0.7 mL of dilute hydrochloric acid of 1 mol/L, adds 0.8 mL of distilled water, 0 ~ 5 DEG C of low temperature stirs
20 min are mixed, 0.1 mL of aqueous solution containing 1.3 mg sodium nitrites is added, continues to stir 1 h, obtains A liquid；60 mg OVA are taken,
0.1 mol/L sodium carbonate liquor, 6 mL is added to dissolve, 0 ~ 5 DEG C of stirring 20 min of equilibrium temperature obtains B liquid, A drop is added to B liquid
In, the reaction was continued 2 h.Stopping reaction, 0.02 mol/L PBS, 3 d of dialysis change liquid three times daily, dispense, obtain coating antigen ,-
20 DEG C of preservations, it is spare.
In the ratio of Metalaxyl synthesizing coupled antigen reaction haptens used, carrier protein and coupled product, carry out ultraviolet
(200 ~ 400 nm) sweep measuring calculates its combination in the absorbance value of 260 nm and 280 nm respectively by comparing three
Than.The maximum absorption band of the maximum absorption band of conjugate metalaxyl hapten-carrier albumen and metalaxyl haptens, carrier protein
Compared to apparent variation has occurred, show that the synthesis of metalaxyl hapten-carrier albumen is successful.Be computed, haptens with
The combination ratio of BSA is 13:1, and the combination ratio of OVA is 10:1.
3, the preparation of metalaxyl monoclonal antibody
1) acquisition of hybridoma
First immunisation: metalaxyl haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is fully emulsified, skin
The Balb/c mouse of 6 week old of lower injection, every 0.2 mL；
Booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, replaces Freund complete with not formula Freund's incomplete adjuvant
Full adjuvant, method and the same first immunisation of dosage；
Potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reach 1:10000 with
Following final immunization was carried out when upper: 0.1 mL of immunogen solution of any adjuvant is not added in intraperitoneal injection, puts to death mouse after three days, takes
Its spleen is merged with myeloma cell；
Cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Using limiting dilution assay to sun
Property hole carry out cloning, obtain and establish the hybridoma cell strain of stably excreting metalaxyl monoclonal antibody, take raw in logarithm
Cell suspension is made with frozen stock solution in long-term hybridoma, is sub-packed in cryopreservation tube, saves for a long time in liquid nitrogen.
2) preparation of monoclonal antibody
Cell recovery: taking out metalaxyl monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt, from
After the heart removes frozen stock solution, culture culture in glassware is moved into；
Preparation ascites and antibody purification: using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing paraffin oil
Only, hybridoma 5 × 10 is injected intraperitoneally in 0.5 mL/ after 7 days5A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate
Method is purified, and metalaxyl monoclonal antibody solution (- 20 DEG C of preservations) is obtained.
3) measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(100000 ~ 300000).
Indirect competitive ELISA method: using metalaxyl haptens-OVA conjugate coated elisa plate, and metalaxyl standard items are added
The sheep anti mouse antiantibody solution of solution, metalaxyl monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min pours out liquid in hole, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of 15 min of reaction are added
Afterwards, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at 450 nm of wavelength.
4) measurement of monoclonal antibody specificity
Antibody specificity refer to the ability of its homospecificity antigen binding compared with such antigen-analogues ability, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
This experiment by metalaxyl and the compound similar with its structure (propachlor, alachlor, Acetochlor, isopropyl methoxalamine,
Pretilachlor, butachlor) it is serially diluted, indirect competitive ELISA is carried out with monoclonal antibody respectively, makes standard curve, analysis
Obtain IC50, cross reacting rate is then calculated as follows:
The cross reacting rate of metalaxyl and its analogue as the result is shown are as follows: metalaxyl 100%, propachlor < 1%, alachlor <
1%, Acetochlor < 1%, isopropyl methoxalamine < 1%, pretilachlor < 1%, butachlor < 1%.Antibody of the present invention to propachlor, alachlor,
The compound no cross reaction similar with metalaxyl structure such as Acetochlor, isopropyl methoxalamine, pretilachlor, butachlor, just for first
White spirit has specific binding.
4, 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, obtains sheep anti mouse antiantibody.
5, metalaxyl monoclonal antibody-colloid gold label object preparation
1) preparation of colloidal gold
The chlorauric acid solution that mass fraction is 1% is diluted to 0.01% with double distilled deionized water, 100 mL 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 the lemon that 1.5 mL mass fractions are 1%
Sour three sodium solutions, continuation are at the uniform velocity heated with stirring to solution in stopping when bright claret, use deionized water after being cooled to room temperature
It is restored to original volume, 4 DEG C of preservations.The colloidal gold prepared detect by an unaided eye be it is clear and transparent, without muddiness, liquid surface without
Floating material, the color of observing colloid gold is claret in the sunlight.
2) metalaxyl monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, it is marked with the pH value of the pH value of 0.2 mol/L solution of potassium carbonate tune colloidal gold to 7.2(different antibodies
Range can change between 7 ~ 8), it is molten to colloidal gold by the standard that 20 ~ 50 μ g antibody are added in every milliliter of colloidal gold solution
Above-mentioned metalaxyl monoclonal antibody is added in liquid, stirs and evenly mixs, is stored at room temperature 10 min, 10% BSA, which is added, makes it in colloidal gold
Whole mass fraction in solution is 1%, stands 10 min.12000 r/min, 4 DEG C of 40 min of centrifugation abandon supernatant, and precipitating is with again
Molten buffer washes twice, with the redissolution buffer that volume is initial colloid gold volume 1/10 will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer: the mass fraction containing BSA be the mass fraction of 0.1% ~ 0.3%, Tween-80 be 0.05% ~ 0.2%,
The 0.02 mol/L phosphate buffer that pH value is 7.2.
6, the preparation of conjugate release pad
Conjugate release pad is soaked in containing 0.5% BSA(mass fraction), pH value be 7.2 0.5 mol/L phosphate buffer
In, 1 h is uniformly soaked, 37 DEG C of 3 h of baking are spare.Film instrument is sprayed by the metalaxyl prepared monoclonal antibody-colloidal gold with Isoflow
For marker even application in conjugate release pad, every 1 cm conjugate release pad sprays 0.01 mL metalaxyl monoclonal antibody-
It after colloid gold label object, is taken out after being placed in 37 DEG C of environment (humidity < 20%) 60 min, is placed in dry environment (humidity < 20%)
In save backup.
7, the preparation of reaction film
Detection line will be constituted in metalaxyl haptens-ovalbumin conjugate coating to reaction film, sheep anti mouse antiantibody is coated with
Nature controlling line is constituted on reaction film.
Coating process: being diluted to 1 mg/mL for metalaxyl haptens-ovalbumin conjugate with phosphate buffer, uses
Isoflow point film instrument is coated in the detection line (T) on nitrocellulose filter, and package amount is 1.0 μ L/cm；With 0.01
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by mol/L, the phosphate buffer that pH value is 7.4, will with Isoflow point film instrument
It is coated in the nature controlling line (C) on nitrocellulose filter, and package amount is 1.0 μ L/cm.The reaction film being coated with is placed in 37 DEG C
Under the conditions of dry 2 h, it is spare.
8, the preparation of sample absorption pad
Sample absorption pad is used containing 0.5% bovine serum albumin(BSA) (mass fraction), the 0.1 mol/L phosphate-buffered that pH value is 7.2
Liquid impregnates 2 h, and it is spare to dry 2 h at 37 DEG C.
9, 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；Conjugate is released
Put 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, instead
The end of film is answered to be connected with the beginning of water absorption pad, the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, the end of water absorption pad
It is aligned with the end of PVC bottom plate；There are detection line and nature controlling line on the reaction film, detection line (T) and nature controlling line (C) are in and institute
State the perpendicular strip tape of the length of test strips；Detection line is located at the side close to the end of conjugate release pad；Nature controlling line is located at
The side of end far from conjugate release pad；Test strips are cut into the small item of 3 mm wide with machine, mounted in special plastics system
In card, it can be reserved for 12 months under the conditions of 4 ~ 30 DEG C.
The detection of metalaxyl in 2 sample of embodiment
1, the pre-treatment of sample
1) cured tobacco leaf sample-pretreating method: the sample of 1.0 ± 0.05 g crushing is weighed into 50 mL centrifuge tubes, is added 10
50% methanol aqueous solution of mL is sufficiently smashed it with refiner；The sample smashed is transferred in syringe, is carried out using filter membrane
Filtering；The filtered sample liquid of 100 μ L is taken, 900 μ L deionized waters are added, is mixed, it is to be checked.
2) fresh tobacco leaf sample-pretreating method: the sample of 2.0 ± 0.05 g crushing is weighed into 50 mL centrifuge tubes, is added
10 mL, 50% methanol aqueous solution is sufficiently smashed it with refiner；The sample smashed is transferred in syringe, filter membrane is used
It is filtered；The filtered sample liquid of 100 μ L is taken, 300 μ L deionized waters are added, is mixed, it is to be checked.
2, it is detected with test strips
70 μ L of sample to be examined solution is taken vertically to drip in well with pipettor, liquid starts timing, reaction 10 when flowing
Min determines result.
3, analysis detection result
Negative (-): the colour developing of T line develops the color deep or develops the color unanimously with C line than C line, indicates that metalaxyl concentration is limited lower than detection in sample,
Such as Fig. 2 a, 2b.
Positive (+): the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color, and indicates that metalaxyl concentration is equal to or higher than in sample
Detection limit, such as Fig. 2 c, 2d.
It is invalid: not occur C line, show that incorrect operating process or test strips have been failed, such as Fig. 2 e, 2f.In this situation
Under, specification should be read over again, and is retested with new test strips.
3 sample detection example of embodiment
1, detection limit test
Blank cured tobacco leaf sample is taken, adding metalaxyl to final concentration wherein is respectively 1,2,4 mg/kg；Take blank fresh tobacco leaf
Sample, adding metalaxyl to final concentration wherein is respectively 0.25,0.5,1.0 mg/kg, takes test strips to be detected, each sample
Originally it is repeated three times.
When detecting cured tobacco leaf sample with test strips, when wherein metalaxyl addition concentration is 1 mg/kg, shown in test strips
The colour developing of T line is shown to develop the color deep than C line or develop the color unanimously with C line, is negative；When wherein metalaxyl addition concentration is 2,4 mg/kg
When, it shows that the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color in test strips, is positive, shows this test strips to cured tobacco leaf
The detection of middle metalaxyl is limited to 2 mg/kg；When detecting fresh tobacco leaf sample with test strips, when wherein metalaxyl addition concentration is 0.25
It shows that the colour developing of T line develops the color deep or develops the color unanimously with C line than C line when mg/kg, in test strips, is negative；When wherein metalaxyl adds
When to add concentration be 0.5,1.0 mg/kg, show that the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color in test strips, be positive, table
Bright test strips are limited to 0.5 mg/kg to the detection of metalaxyl in fresh tobacco leaf.
2, false positive rate, false negative rate test
Take known cured tobacco leaf positive sample of the metalaxyl content greater than 2 mg/kg and metalaxyl content greater than 0.5 mg/kg's
Each 20 parts of fresh tobacco leaf positive sample, it is known that cured tobacco leaf negative sample and metalaxyl content of the metalaxyl content less than 2 mg/kg
Each 20 parts of fresh tobacco leaf negative sample less than 0.5 mg/kg, is detected with three batches of test strips, calculates its yin and yang attribute rate.
The result shows that: when detecting positive sample with the test strips of 3 batch productions, as a result it is all positive, it is known that positive sample
Product coincidence rate is 100%, false negative rate 0；When detecting negative sample, as a result it is all negative, it is known that negative sample coincidence rate is
100%, false positive rate 0.Illustrate that the test strips of detection metalaxyl of the invention can quickly examine the metalaxyl in tobacco leaf
It surveys.
3, specific test
It is with pH value by the metalaxyls analogue such as propachlor, alachlor, Acetochlor, isopropyl methoxalamine, pretilachlor, butachlor
7.2, the phosphate buffer of 0.2 mol/L is diluted to 50 mg/L, is detected with metalaxyl test strips.The results show that with should
When test strips detect 50 mg/L propachlors, alachlor, Acetochlor, isopropyl methoxalamine, pretilachlor, butachlor, test strips T line is aobvious
The colour developing of color ratio C line is deep or consistent with the colour developing of C line, is negative.Illustrate this test strips to propachlor, alachlor, Acetochlor, isopropyl first
The metalaxyls analogue no cross reaction such as careless amine, pretilachlor, butachlor.
Claims (6)
1. a kind of test strips for detecting metalaxyl, including sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；
It is characterized by: there is the detection line for being coated with metalaxyl hapten-carrier protein conjugate on the reaction film and be coated with
The nature controlling line of sheep anti mouse antiantibody is coated with metalaxyl monoclonal antibody-colloid gold label object in the conjugate release pad；Institute
Stating metalaxyl monoclonal antibody is prepared using metalaxyl hapten-carrier protein conjugate as immunogene, the first frost
Clever hapten-carrier protein conjugate is obtained by metalaxyl haptens with carrier protein couplet, and the carrier protein is cow's serum
Albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumins, the metalaxyl haptens are by N- (2,6-
3,5-dimethylphenyl) methyl lactamine and (4-nitrophenoxy) excess acetyl chloride generate nitre phenyl metalaxyl, then also by zinc powder
Original obtains, molecular structural formula are as follows:
。
2. test strips according to claim 1, it is characterised in that: the preparation reaction process of the metalaxyl haptens is such as
Under:
。
3. test strips according to claim 1, it is characterised in that: the sample absorption pad, conjugate release pad, reaction
Film, water absorption pad are successively pasted on bottom plate, and conjugate release pad 1/3 ~ 1/2 is capped under sample absorption pad.
4. test strips according to claim 1, it is characterised in that: it is immune that the sheep anti mouse antiantibody, which is with source of mouse antibody,
Original carries out immune prepare to goat.
5. a kind of method for preparing any one of claim 1-4 test strips, it is characterised in that: the following steps are included:
1) preparation is coated with metalaxyl monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with metalaxyl hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
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.
6. a kind of method using metalaxyl in any one of the claim 1-4 test strips detection tobacco sample, feature exist
In: method includes the following steps:
1) pre-treatment is carried out to sample；
2) it is detected with the test strips；
3) analysis detection result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104762.0A CN109061155B (en) | 2018-09-21 | 2018-09-21 | Test strip for detecting metalaxyl and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104762.0A CN109061155B (en) | 2018-09-21 | 2018-09-21 | Test strip for detecting metalaxyl and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109061155A true CN109061155A (en) | 2018-12-21 |
| CN109061155B CN109061155B (en) | 2021-05-11 |
Family
ID=64763297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811104762.0A Active CN109061155B (en) | 2018-09-21 | 2018-09-21 | Test strip for detecting metalaxyl and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061155B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232464A (en) * | 1996-07-31 | 1999-10-20 | 拜尔公司 | Substd. N-isothiazolyl-(thio) amides |
| US20010026920A1 (en) * | 2000-02-25 | 2001-10-04 | Mark Chandler | Internal standards and controls for multiplexed assay |
| CN1680405A (en) * | 2005-01-21 | 2005-10-12 | 浙江大学 | Chlorpyrifos artificial semiantigen, antigen, antibody and use, synthesis of semiantigen |
| CN101093196A (en) * | 2007-07-25 | 2007-12-26 | 中华人民共和国天津出入境检验检疫局 | Meter for reading test paper tape of colloidal gold in use for detecting residue |
| WO2014086851A1 (en) * | 2012-12-04 | 2014-06-12 | Basf Agro B.V., Arnhem (Nl) | Compositions comprising a quillay extract and a fungicidal inhibitor of respiratory complex iii at qo site |
| CN104569399A (en) * | 2013-10-22 | 2015-04-29 | 北京勤邦生物技术有限公司 | Test strip for testing ochratoxin A and application of test strip |
| CN105259351A (en) * | 2015-08-26 | 2016-01-20 | 贵州勤邦食品安全科学技术有限公司 | Test paper for detecting metalaxyl residue and application thereof |
| CN106543049A (en) * | 2016-10-25 | 2017-03-29 | 中国农业大学 | The preparation method of sulfonamides hapten and its conjugated antigen and application |
| WO2018057647A1 (en) * | 2016-09-23 | 2018-03-29 | Alveo Technologies, Inc. | Methods and compositions for detecting analytes |
| WO2018087990A1 (en) * | 2016-11-10 | 2018-05-17 | 東レ株式会社 | Diamine compound, heat-resistant resin using same, and resin composition |
-
2018
- 2018-09-21 CN CN201811104762.0A patent/CN109061155B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232464A (en) * | 1996-07-31 | 1999-10-20 | 拜尔公司 | Substd. N-isothiazolyl-(thio) amides |
| US20010026920A1 (en) * | 2000-02-25 | 2001-10-04 | Mark Chandler | Internal standards and controls for multiplexed assay |
| CN1680405A (en) * | 2005-01-21 | 2005-10-12 | 浙江大学 | Chlorpyrifos artificial semiantigen, antigen, antibody and use, synthesis of semiantigen |
| CN101093196A (en) * | 2007-07-25 | 2007-12-26 | 中华人民共和国天津出入境检验检疫局 | Meter for reading test paper tape of colloidal gold in use for detecting residue |
| WO2014086851A1 (en) * | 2012-12-04 | 2014-06-12 | Basf Agro B.V., Arnhem (Nl) | Compositions comprising a quillay extract and a fungicidal inhibitor of respiratory complex iii at qo site |
| CN104569399A (en) * | 2013-10-22 | 2015-04-29 | 北京勤邦生物技术有限公司 | Test strip for testing ochratoxin A and application of test strip |
| CN105259351A (en) * | 2015-08-26 | 2016-01-20 | 贵州勤邦食品安全科学技术有限公司 | Test paper for detecting metalaxyl residue and application thereof |
| WO2018057647A1 (en) * | 2016-09-23 | 2018-03-29 | Alveo Technologies, Inc. | Methods and compositions for detecting analytes |
| CN106543049A (en) * | 2016-10-25 | 2017-03-29 | 中国农业大学 | The preparation method of sulfonamides hapten and its conjugated antigen and application |
| WO2018087990A1 (en) * | 2016-11-10 | 2018-05-17 | 東レ株式会社 | Diamine compound, heat-resistant resin using same, and resin composition |
Non-Patent Citations (6)
| Title |
|---|
| NEWSOME,WH: "An Enzyme-Linked Immunosorbent Assay for Metalaxyl in Foods", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| SATHE,MANISHA 等: "Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): An organophosphorous toxicant", 《JOURNAL OF HAZARDOUS MATERIALS》 * |
| 刘学东 等: "甲霜灵及其旋光体的合成进展", 《农药》 * |
| 周洪斌 等: "进出口植物产品中甲霜灵残留量酶联免疫检测试剂盒的研究", 《免疫学杂志》 * |
| 李俊锁 等: "磺胺类药物半抗原的合成及结构表征", 《中国农业大学学报》 * |
| 赵友全 等: "一种新型便携式甲霜灵胶体金试纸条显色分析仪的研制", 《仪器仪表学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109061155B (en) | 2021-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104569399B (en) | A kind of test strips detecting ochratoxin A and application thereof | |
| CN105424939B (en) | A kind of test strips for detecting carbendazim and its preparation method and application | |
| CN105675874B (en) | A kind of colloidal gold strip for detecting imidacloprid and its application | |
| CN108776219B (en) | Immunochromatographic test strip for rapidly detecting alternaria tenuipili acid | |
| CN105807055B (en) | A kind of test strips for detecting dichloro quinolinic acid and its preparation method and application | |
| CN109265401A (en) | A kind of preparation method and application of iprodione haptens and antigen | |
| CN109265404A (en) | A kind of preparation method and application of carbendazim haptens and antigen | |
| CN108196054A (en) | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application | |
| CN105785011B (en) | A kind of test strips for detecting maleic hydrazide and its preparation method and application | |
| CN109061146A (en) | A kind of test strips and its preparation method and application detecting Acetamiprid | |
| CN110441512A (en) | A kind of colloidal gold immunochromatographimethod detection device of ethylmaltol haptens and ethylmaltol | |
| CN110441518A (en) | A kind of test strips and method detecting gibberellin | |
| CN109239336A (en) | A kind of test strips and its application detecting Mobucin | |
| CN109061147A (en) | A kind of test strips and its preparation method and application detecting pendimethalin | |
| CN109917126A (en) | A method of test strips, the preparation method of imidacloprid haptens and the detection Determination of Imidacloprid Residue of detection imidacloprid | |
| CN105624119B (en) | The general monoclonal antibody of anti-capsaicine, Dihydrocapsaicin, the synthetic capsaicin of hybridoma cell strain YQQD8 and its generation | |
| CN103364546B (en) | A kind of kit and method detecting Furaxone metabolite | |
| CN112250641A (en) | Hydrochlorothiazide hapten, artificial antigen, antibody and preparation method and application thereof | |
| CN108931640B (en) | Test strip for detecting organophosphorus pesticide and application thereof | |
| CN111500546A (en) | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card | |
| CN103940999B (en) | A kind of test strip detecting Betamethasone Valerate and its preparation method and application | |
| CN109061155A (en) | A kind of test strips and its preparation method and application detecting metalaxyl | |
| CN109061148A (en) | A kind of test strips and its preparation method and application detecting butralin | |
| CN108997161A (en) | A kind of preparation method and application of metalaxyl haptens and antigen | |
| CN109061145A (en) | A kind of test strips and its preparation method and application detecting flumetralim |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |