CN109336779A - A kind of preparation and application of metalaxyl haptens and antigen - Google Patents

A kind of preparation and application of metalaxyl haptens and antigen Download PDF

Info

Publication number
CN109336779A
CN109336779A CN201811104602.6A CN201811104602A CN109336779A CN 109336779 A CN109336779 A CN 109336779A CN 201811104602 A CN201811104602 A CN 201811104602A CN 109336779 A CN109336779 A CN 109336779A
Authority
CN
China
Prior art keywords
metalaxyl
antigen
haptens
preparation
reaction
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
Application number
CN201811104602.6A
Other languages
Chinese (zh)
Other versions
CN109336779B (en
Inventor
陈黎
范子彦
刘惠民
唐纲岭
赵乐
崔华鹏
樊美娟
鲁亚辉
朱亮
冯才伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Kwinbon Biotechnology Co Ltd
Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
Original Assignee
Beijing Kwinbon Biotechnology Co Ltd
Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beijing Kwinbon Biotechnology Co Ltd, Zhengzhou Tobacco Research Institute of CNTC, National Tobacco Quality Supervision and Inspection Center filed Critical Beijing Kwinbon Biotechnology Co Ltd
Priority to CN201811104602.6A priority Critical patent/CN109336779B/en
Publication of CN109336779A publication Critical patent/CN109336779A/en
Application granted granted Critical
Publication of CN109336779B publication Critical patent/CN109336779B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/16—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/577—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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A kind of preparation and application of metalaxyl haptens and antigen, it is characterised in that: the metalaxyl haptens is to carry out substitution reaction under the catalysis of aluminium chloride by metalaxyl and 4- aldehyde radical butyl chloride to obtain；The metalaxyl antigen is to be obtained by metalaxyl haptens with carrier protein couplet.Antigen prepared by the present invention shows the metalaxyl antigenic determinant of specificity, makes it possible the metalaxyl monoclonal antibody for filtering out high specific.The antibody specificity height of generation, high sensitivity, can be used for establishing enzyme-linked immunosorbent assay for measuring and colloid gold test paper rapid test method, to realize the quick detection of metalaxyl in tobacco and food.

Description

A kind of preparation and application of metalaxyl haptens and antigen
Technical field
The present invention relates to the preparations and application of a kind of metalaxyl haptens and antigen.Belong to pesticide immunochemical technique neck Domain.
Background technique
Metalaxyl (Metalaxyl) is amides systemic fungicide, has preventive and therapeutic action, for preventing and treating melon Fruits and vegetables downy mildew, epidemic disease, potato morning, late blight, tobacco black shank etc..Although metalaxyl is not high to the toxicity of people and animals, Belong to absorbability sterilization, the seasonal exceeded situation of the residual quantity in food and vegetables is more.China is directed to Different Crop, formulates The maximum residue limit standard of metalaxyl, wherein cucumber, capsicum, tomato maximum residue limit be 0.5 mg/kg, brown rice Maximum residue limit is 0.1 mg/kg, and the maximum residue limit of other cereal is 0.05 mg/kg.The U.S., Canada, Japan, The states such as European Union have formulated the maximum residue limit of metalaxyl in national plant product.International tobacco scientific research Cooperation Centre (CORESTA) the guiding residue limits for providing metalaxyl in tobacco are 2 mg/kg.
Currently, the method for metalaxyl residue detection mainly has the instruments sides such as LC-UV, LC-MS, LC-MS/MS, GC-MS/MS Method, traditional instrument analysis method has the advantages such as detection sensitivity height, high specificity, but sample pre-treatments are cumbersome, time-consuming, simultaneously Detection needs expensive large-scale instrument and equipment, and need to be equipped with the detection technique personnel of profession, can not carry out live extensive inspection It surveys, poor in timeliness, it is difficult to promote.Immunoassay method based on antigen and antibody specific identification can be with qualitative and quantitative detection sample In pesticide residue.This analysis method is of less demanding to instrument and equipment, fast and convenient, general without carrying out complexity to sample Pretreatment, high sensitivity, high specificity are of less demanding to the professional technique of user of service, are easy universal and promote, and can meet fast The needs of fast analysis detection are particularly suitable for the quick analysis of scene screening and a large amount of samples.Immunoassay grinds for metalaxyl residue Study carefully and provides a new analysis detection approach.Immunoassay has become a brand-new neck of pesticide residue analysis research at present Immunoassay and gas-chromatography, liquid chromatogram are classified as three big mainstays of pesticide residue analysis by domain, American Chemical Society jointly. China's pesticide immuno analytical method research starts to walk relatively late, but quickly grows in recent years, about parathion, methyl to sulphur Phosphorus, methyl paraoxon, carbendazim, chlopyrifos, Hostathion, Fipronil, dichloro quinolinic acid, carbofuran, triazolone, acephatemet, Ah The preparation of the specific antibody of the artificial antigen and high-affinity of the pesticides such as Te Lajin, 2 first, 4 chlorine and sample is carried out with enzyme-linked immunization The report of the trace analysis of agricultural drugs in product.
The invention belongs to pesticide molecule compound immunochemistries and retention analysis technical field, are related to organic synthesis, exempt from Epidemic disease chemistry and biochemistry etc., by immunology, immunochemistry basic principle and biotechnological method, design, synthesized micromolecule Target analytes haptens, and be coupled with carrier protein, prepare effective artificial antigen.The antigen of preparation can be by immune dynamic Object prepares the antibody to small molecule analyte specific recognition, is detected using the specificity immunology reaction of antigen-antibody with easy The amplification of the marker of identification, ultramicron small molecule object in quantitative test sample.The MOLECULE DESIGN of haptens with Synthesis is the committed step for generating specific antibody and establishing pesticide residue immunoassay method.The preparation of artificial antigen, including Difference on binding site, combination, carrier and haptens and any structure of analyte substance of interest, such as molecule are big Topological sex character including small, shape, ingredient, configuration, conformation, polarity, cloud density etc., all may strong influence it is corresponding The property of antibody.The better haptens of performance, effect and antigen can be designed, emphasis of interest exactly of the invention.
Summary of the invention
The purpose of the present invention is based on above-mentioned prior art situation and provides the system of a kind of metalaxyl haptens and antigen Preparation Method.
The purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of metalaxyl haptens is to be taken under the catalysis of aluminium chloride by metalaxyl with 4- aldehyde radical butyl chloride Generation reaction obtains, molecular structural formula are as follows:
。
Specific step is as follows:
0.53 g of 4- aldehyde radical butyl chloride is taken, 30 mL 1 are added, the dissolution of 2- dichloroethanes adds 0.44 g aluminium chloride, is stirred at room temperature 3 H, is added dropwise 1,2- dichloroethane solution, 20 mL containing 0.6 g metalaxyl, and 4 h of back flow reaction is cooled to room temperature, adds 40 mL Water, 0.5 mL dilute hydrochloric acid, oscillation layering divide and go water phase, and organic phase saturated common salt water washing is dry, concentration, and upper silicagel column is used The petroleum ether-ethyl acetate that volume ratio is 1:1 elutes separation, obtains -4 aldehyde Butyrylation metalaxyl 0.71 of metalaxyl haptens product g。
The metalaxyl haptens can be used for making the antigen system raw material of animal immune.
A kind of preparation method of metalaxyl antigen is to be obtained by the metalaxyl haptens with carrier protein couplet.It is described Carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or human serum albumins.
Specific step is as follows:
The preparation of immunizing antigen: taking 16 mg haptens, is dissolved in 1 mL n,N-Dimethylformamide (DMF), stirring clarification, It is denoted as reaction solution A；30 mg of bovine serum albumin(BSA) (BSA) is weighed, is allowed to be substantially dissolved in 2.7 mL, 0.1 mol/L citrate In buffer (CB, pH value 9.6), reaction solution A is slowly dropped in protein solution dropwise, and stir 24 h at room temperature, With 0.5 mL reduction reaction of sodium borohydride aqueous solution, 4 h of 3 M, dialysed with 4 DEG C of 0.01 mol/L phosphate buffer (PBS) 3 d change 3 dialyzates daily to remove unreacted small-molecule substance and obtain immunogene.
The preparation of envelope antigen: taking 12 mg haptens, is dissolved in 1 mL DMF and dissolves clarification, is denoted as reaction solution A；Claim Take 30 mg of ovalbumin (OVA), be allowed to be substantially dissolved in 2.7 mL, 0.1 mol/L CB(pH value be 9.6) in, by reaction solution A It is slowly dropped in protein solution dropwise, and stirs 24 h at room temperature, restored with 0.5 mL of sodium borohydride aqueous solution of 3 M 4 h are reacted, with 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis, change 3 dialyzates, daily to remove unreacted small molecule object Matter obtains coating antigen.
The application of the metalaxyl antigen of preparation: the monoclonal antibody obtained using the metalaxyl antigen-immunized animal, it can For quickly detecting the metalaxyl residue in tobacco and food.
The metalaxyl haptens and antigen that are synthesized in the present invention with application No. is 201510529378.5 and 201510530296.2 patent in haptens and antigenic structure it is different.The metalaxyl haptens synthesized in the present invention was both maximum Degree remains the chemical structure of metalaxyl, and has the linking arm of appropriate length, goes to exempt from immunogene prepared by the haptens Epidemic disease animal, potency, specificity, the affinity of obtained antibody are all relatively good, low with the cross reacting rate of other pesticides, specificity More preferably, sensitivity is higher.
Antigen prepared by the present invention shows the metalaxyl antigenic determinant of specificity, so that filtering out the first of high specific White spirit monoclonal antibody is possibly realized.The antibody specificity height of generation, high sensitivity, can be used for establishing enzyme linked immunosorbent assay (ELISA) Method and colloid gold test paper rapid test method, to realize the quick detection of metalaxyl in tobacco and food.
Detailed description of the invention
Fig. 1 is metalaxyl hapten synthesis route map (figure is Figure of abstract)；
Fig. 2 is test paper the schematic diagram of the section structure, in figure: 1, sample absorption pad；2, reaction film；3, water absorption pad；4, detection line；5, matter Control line；6, bottom plate；7, protective film；
Fig. 3 is test paper top view；
Fig. 4 is micropore reagent figure, in figure: 8, micropore；9, micropore plug.
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.
The preparation of 1 metalaxyl haptens of embodiment
1, the synthesis of metalaxyl haptens (synthetic route is shown in attached drawing 1)
0.53 g of 4- aldehyde radical butyl chloride is taken, 30 mL 1 are added, the dissolution of 2- dichloroethanes adds 0.44 g aluminium chloride, is stirred at room temperature 3 H, is added dropwise 1,2- dichloroethane solution, 20 mL containing 0.6 g metalaxyl, and 4 h of back flow reaction is cooled to room temperature, adds 40 mL Water, 0.5 mL dilute hydrochloric acid, oscillation layering divide and go water phase, and organic phase saturated common salt water washing is dry, concentration, and upper silicagel column is used The petroleum ether-ethyl acetate that volume ratio is 1:1 elutes separation, obtains -4 aldehyde Butyrylation metalaxyl 0.71 of metalaxyl haptens product G, yield 89.7%.
2, the identification of metalaxyl haptens
Above-mentioned haptens is taken to identify through nuclear magnetic resonance spectroscopy,1H NMR(CDCl3, 300 HZ) δ: 4.735 (4,1H, q, J= 7.072), 3.661 (5,3H), 1.403 (7,3H, d, J=7.072), 4.046 (13,2H), 2.351 (15,3H), 7.367 (16,1H, d, J=8.304), 2.110 (17,3H), 7.011 (20,1H, d, J=8.304), 3.206 (21,3H), 2.608 (23,2H, t, J=7.426), 2.944 (24,2H, td, J=7.426, J=6.840), 9.659 (25,1H, t, J=6.840).
Chemical shift δ=2.9 in map, 2.6 be methylene hydrogen on spacerarm resonance absorbing peak, δ=9.6 are intervals Aldehyde radical resonance absorbing peak on arm, the presence of the absorption peak of these hydrogen, it was demonstrated that hapten synthesis success.
The preparation of 2 metalaxyl antigen of embodiment
1, the preparation of metalaxyl immunogene
Metalaxyl haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
16 mg haptens are taken, are dissolved in 1 mL n,N-Dimethylformamide (DMF), stirring clarification is denoted as reaction solution A；30 mg of BSA is weighed, is allowed to be substantially dissolved in 2.7 mL, 0.1 mol/L citrate buffer (CB, pH value 9.6), Reaction solution A is slowly dropped in protein solution dropwise, and stirs 24 h at room temperature, with the sodium borohydride aqueous solution of 3 M 0.5 mL reduction reaction, 4 h changes 3 dialyzates with 0.01 mol/L phosphate buffer (PBS), 4 DEG C of 3 d of dialysis daily, with Unreacted small-molecule substance is removed, immunogene is obtained.
2, the preparation of metalaxyl coating antigen
Metalaxyl haptens and ovalbumin (OVA) coupling obtain coating antigen.
12 mg haptens are taken, is dissolved in 1 mL DMF and dissolves clarification, be denoted as reaction solution A；30 mg of OVA is weighed, is allowed to Be substantially dissolved in 2.7 mL, 0.1 mol/L CB(pH value be 9.6) in, reaction solution A is slowly dropped in protein solution dropwise, And 24 h are stirred at room temperature, with 0.5 mL reduction reaction of sodium borohydride aqueous solution, 4 h of 3 M, with 0.01 mol/L PBS 4 DEG C dialysis 3 d, change 3 dialyzates daily to remove unreacted small-molecule substance and obtain coating antigen.
3, the identification of metalaxyl antigen
In the ratio of Metalaxyl synthesizing coupled antigen reaction haptens used, carrier protein and coupled product, ultraviolet (200 are carried out ~ 400 nm) sweep measuring, by comparing three respectively the absorbance value of 260 nm and 280 nm calculate its combine than.Coupling The maximum absorption band of object metalaxyl hapten-carrier albumen with metalaxyl haptens, carrier protein maximum absorption band compared with send out Apparent variation has been given birth to, has shown that the synthesis of metalaxyl hapten-carrier albumen is successful.It is computed, the knot of haptens and BSA Composition and division in a proportion is 18:1, and the combination ratio of OVA is 13:1.
The preparation of 3 metalaxyl monoclonal antibody of embodiment
1, animal immune
Obtained immunogene is injected in Balb/c Mice Body, immunizing dose is 150 μ g/, its is made to generate antiserum.
2, cell fusion and cloning
Immune Balb/c mouse boosting cell being taken, in 8:1(quantitative proportion) ratio merges with SP2/0 myeloma cell, and screening obtains The metalaxyl monoclonal antibody hybridoma cell strain of stably excreting metalaxyl monoclonal antibody.
3, cell cryopreservation and recovery
Metalaxyl monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, centrifugation removal is frozen After liquid storage, culture culture in glassware is moved into.
4, the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, with octanoic acid-saturation Ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture medium, and calf serum is made to exist Final concentration of 20%(mass fraction in cell culture medium), the final concentration of 0.2%(mass of sodium bicarbonate in cell culture medium Score)；The pH value of the cell culture medium is 7.4.
5, the measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(200000 ~ 500000).
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.
6, the 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.
The preparation of 4 metalaxyl colloidal gold strip of embodiment
1, 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.Preparing good colloidal gold and detecting by an unaided eye is clear and transparent, no muddiness, liquid table Face is without floating material, and 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 used Redissolve buffer wash twice, with the redissolution buffer that volume is initial colloid gold volume 1/10 will precipitating be resuspended, set 4 DEG C it is standby With.
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.
2, the preparation of micropore reagent
100 μ L metalaxyl monoclonal antibodies-colloid gold label object is added into micropore reagent micropore, is put into freeze drier, Under the conditions of condenser temperature is -50 DEG C, after 3 h of pre-freeze, then 15 h is dried in vacuo, that is, can be taken off, obtaining freeze-drying has metalaxyl list Clonal antibody-colloid gold label object micropore reagent, is sealed.
3, the preparation of sample absorption pad
It is 0.5% bovine serum albumin(BSA), the 0.1 mol/L phosphate-buffered that pH value is 7.2 that sample absorption pad, which is placed in volume fraction, 2h is impregnated in liquid, 37 DEG C of baking 2h are spare.
4, the preparation of reaction film
Coating process: metalaxyl haptens-ovalbumin conjugate is diluted to 1 mg/mL 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 0.01mol/L, pH value Sheep anti mouse antiantibody is diluted to 200 μ g/mL for 7.4 phosphate buffer, is coated in nitre with Isoflow point film instrument Nature controlling line (C) on acid cellulose film, package amount are 1.0 μ L/cm.It is dry under the conditions of the reaction film being coated with is placed in 37 DEG C 2h, it is spare.
5, the assembling of each component
(1) assembling of test paper
The sample absorption pad, reaction film, water absorption pad are successively pasted on the bottom plate in order；The end of sample absorption pad It is connected with the beginning of reaction film, the end of reaction film is connected with the beginning of water absorption pad, the beginning of sample absorption pad and the beginning of bottom plate End alignment, the end of water absorption pad is aligned with the end of bottom plate；The bonding protective film on the sample absorption pad of assembled test paper is protected MAX mark line is printed on cuticula.
(2) assembling of test strips
Test paper and micropore reagent that above-mentioned steps 1 obtain are assembled into test strips, stored in 2 ~ 8 DEG C of environment, validity period 12 A month.
Embodiment 5 detects the application of the colloidal gold strip of metalaxyl
1, the pre-treatment of sample
The smashed sample to be tested of 1.0 ± 0.05 g is weighed into 50 mL centrifuge tubes, 10 mL, 50% methanol aqueous solution, whirlpool is added 1 min is revolved, 3000 rpm or more are centrifuged 5 min；100 μ L supernatants are taken, 400 μ L samples are added and redissolve liquid, mix to be measured.
2, it is detected with test strips
With micropipettor draw 100 μ L sample to be tested solution in micropore reagent, slowly suction and sufficiently with reagent in micropore It mixes, after (20 ~ 25 DEG C) 3 min of incubation of room temperature, test paper is indicated into MAX label line end and is downwardly into the micropore reagent after being incubated for In, liquid starts timing when flowing, react 10 min, determines result according to schematic diagram.
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.
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.
It is invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips.It in the case, should be again It is secondary to read over specification, and retested with new test strips.
Embodiment 6 detects the determination of the colloidal gold strip technical parameter of metalaxyl
1, detection limit test
Take blank agricultural product and tobacco sample, wherein respectively addition metalaxyl to final concentration of 0.05,0.1,0.2 mg/kg, Test strips are taken to be detected, each sample is repeated three times.
When detecting agricultural product and tobacco sample with test strips, when wherein metalaxyl addition concentration is 0.05 mg/kg, examination It shows that the colour developing of T line develops the color deep or develops the color unanimously with C line than C line on paper slip, is negative；When wherein metalaxyl addition concentration is 0.1, 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 when 0.2 mg/kg, in test strips, is positive, shows this test paper Item is limited to 0.1 mg/kg to the detection of metalaxyl in agricultural product.
2, false positive rate, false negative rate test
It takes known metalaxyl content greater than 20 parts of positive sample of 0.1 mg/kg, 20 parts of negative sample, is carried out with three batches of test strips Detection, 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 detection metalaxyl of the invention test strips can to the metalaxyl in agricultural product and tobacco into Row quickly detection.
3, specific test
By other metalaxyl analogues such as propachlor, alachlor, Acetochlor, isopropyl methoxalamine, pretilachlor, butachlor pH Value is that the phosphate buffer of 7.2,0.2 mol/L is diluted to 1 mg/L, is detected with metalaxyl test strips.The results show that When detecting 1 mg/L propachlor, alachlor, Acetochlor, isopropyl methoxalamine, pretilachlor, butachlor with the test strips, test strips T line Colour developing develops the color deep or develops the color unanimously with C line than C line, is negative.Illustrate this test strips to propachlor, alachlor, Acetochlor, isopropyl The compound no cross reaction similar with metalaxyl structure such as alachlor, pretilachlor, butachlor, specificity are good.

Claims (8)

1. a kind of preparation method of metalaxyl haptens, it is characterised in that: be by metalaxyl and 4- aldehyde radical butyl chloride in aluminium chloride Catalysis under carry out substitution reaction and obtain, molecular structural formula are as follows:
。
2. the preparation method of metalaxyl haptens as described in claim 1, it is characterised in that: the specific steps of the preparation method It is as follows:
0.53 g of 4- aldehyde radical butyl chloride is taken, 30 mL 1 are added, the dissolution of 2- dichloroethanes adds 0.44 g aluminium chloride, is stirred at room temperature 3 H, is added dropwise 1,2- dichloroethane solution, 20 mL containing 0.6 g metalaxyl, and 4 h of back flow reaction is cooled to room temperature, adds 40 mL Water, 0.5 mL dilute hydrochloric acid, oscillation layering divide and go water phase, and organic phase saturated common salt water washing is dry, concentration, and upper silicagel column is used The petroleum ether-ethyl acetate that volume ratio is 1:1 elutes separation, obtains -4 aldehyde Butyrylation metalaxyl 0.71 of metalaxyl haptens product g。
3. the application of the metalaxyl haptens of method preparation as described in claim 1, it is characterised in that: the metalaxyl haptens It can be used for making the antigen system raw material of animal immune.
4. a kind of preparation method of metalaxyl antigen, it is characterised in that: be the metalaxyl haptens prepared by claim 1 and load Body protein is coupled to obtain.
5. the preparation method of metalaxyl antigen as claimed in claim 4, it is characterised in that: the carrier protein is that ox blood is pure Albumen, ovalbumin, hemocyanin, thyroprotein or human serum albumins.
6. the preparation method of metalaxyl antigen as described in claim 4 or 5, it is characterised in that: specific step is as follows: taking 16 Mg haptens is dissolved in 1 mL n,N-Dimethylformamide (DMF), and stirring clarification is denoted as reaction solution A；It is pure to weigh ox blood 30 mg of albumen is allowed to be substantially dissolved in 2.7 mL, 0.1 mol/L citrate buffer (CB, pH value 9.6), will react Liquid A is slowly dropped in protein solution dropwise, and stirs 24 h at room temperature, also with 0.5 mL of sodium borohydride aqueous solution of 3 M 4 h of original reaction change 3 dialyzates with 0.01 mol/L phosphate buffer (PBS), 4 DEG C of 3 d of dialysis daily, not anti-to remove The small-molecule substance answered obtains metalaxyl antigen；Packing, -20 DEG C save backup.
7. the preparation method of metalaxyl antigen as described in claim 4 or 5, it is characterised in that: specific step is as follows: taking 12 Mg haptens is dissolved in 1 mL DMF and dissolves clarification, is denoted as reaction solution A；30 mg of ovalbumin is weighed, is allowed to sufficiently dissolve 2.7 mL, 0.1 mol/L CB(pH value be 9.6) in, reaction solution A is slowly dropped in protein solution dropwise, and in room temperature 24 h of lower stirring, with 0.5 mL reduction reaction of sodium borohydride aqueous solution, 4 h of 3 M, with 4 DEG C of 0.01 mol/L PBS dialysis 3 D changes 3 dialyzates daily, to remove unreacted small-molecule substance, obtains metalaxyl antigen；Packing, -20 DEG C save backup.
8. the application of the metalaxyl antigen of method preparation as claimed in claim 4, it is characterised in that: use prepared metalaxyl The monoclonal antibody that antigen-immunized animal obtains, can be used for establishing enzyme-linked immunosorbent assay for measuring and colloid gold test paper is quickly surveyed Method is determined, to realize the quick detection of metalaxyl in tobacco and food.
CN201811104602.6A 2018-09-21 2018-09-21 Preparation and application of metalaxyl hapten and antigen Active CN109336779B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811104602.6A CN109336779B (en) 2018-09-21 2018-09-21 Preparation and application of metalaxyl hapten and antigen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811104602.6A CN109336779B (en) 2018-09-21 2018-09-21 Preparation and application of metalaxyl hapten and antigen

Publications (2)

Publication Number Publication Date
CN109336779A true CN109336779A (en) 2019-02-15
CN109336779B CN109336779B (en) 2021-02-19

Family

ID=65306480

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811104602.6A Active CN109336779B (en) 2018-09-21 2018-09-21 Preparation and application of metalaxyl hapten and antigen

Country Status (1)

Country Link
CN (1) CN109336779B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1945331A (en) * 2006-10-20 2007-04-11 邹明强 Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds
CN105158472A (en) * 2015-08-26 2015-12-16 贵州勤邦食品安全科学技术有限公司 Enzyme linked immunosorbent assay kit for detecting metalaxyl residues and using method
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1945331A (en) * 2006-10-20 2007-04-11 邹明强 Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds
CN105158472A (en) * 2015-08-26 2015-12-16 贵州勤邦食品安全科学技术有限公司 Enzyme linked immunosorbent assay kit for detecting metalaxyl residues and using method
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

Also Published As

Publication number Publication date
CN109336779B (en) 2021-02-19

Similar Documents

Publication Publication Date Title
CN106918704B (en) Synchronous detection aflatoxin and the time-resolved fluoroimmunoassay of carbaryl composite pollution chromatograph kit, preparation method and application
CN100482643C (en) Semicarbazide derivative, monoclonal antibody thereof and application
CN104569399B (en) A kind of test strips detecting ochratoxin A and application thereof
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
CN107907687A (en) A kind of dicofol haptens preparation method and applications
CN109265404A (en) A kind of preparation method and application of carbendazim haptens and antigen
CN110441518B (en) Test strip and method for detecting gibberellin
CN109232286A (en) A kind of preparation method and application of pendimethalin haptens and antigen
CN110441512A (en) A kind of colloidal gold immunochromatographimethod detection device of ethylmaltol haptens and ethylmaltol
CN109324182A (en) A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting pendimethalin
CN109206332A (en) A kind of preparation method and application of flumetralim haptens and antigen
CN105785011B (en) A kind of test strips for detecting maleic hydrazide and its preparation method and application
CN109053477A (en) A kind of preparation method and application of butralin haptens and antigen
CN111735951B (en) Test strip for detecting fenpropathrin and application thereof
CN103288661A (en) Preparation method and application of malachite green hapten
CN109061154A (en) A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting metalaxyl
CN110551220A (en) Preparation and application of DDT monoclonal antibody
CN109336779A (en) A kind of preparation and application of metalaxyl haptens and antigen
CN108459000A (en) Time-resolved fluorescence test strip for detecting fluopyram and application thereof
CN108469517A (en) Time-resolved fluorescent test strip for detecting trichlorfon and application thereof
CN109265364A (en) A kind of preparation and application of pendimethalin haptens and antigen
CN108997161A (en) A kind of preparation method and application of metalaxyl haptens and antigen
CN109265395A (en) A kind of preparation method and application of dichloro quinolinic acid haptens and antigen
CN108196046A (en) A kind of remaining enzyme-linked immune detection method of gibberellin

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