CN105092833A - Oximido acetate bactericide detection ELISA method - Google Patents

Oximido acetate bactericide detection ELISA method Download PDF

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CN105092833A
CN105092833A CN201410200830.9A CN201410200830A CN105092833A CN 105092833 A CN105092833 A CN 105092833A CN 201410200830 A CN201410200830 A CN 201410200830A CN 105092833 A CN105092833 A CN 105092833A
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acetic acid
acid ester
bactericidal agent
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ester series
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戴贤君
朱洁
杨维
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China Jiliang University
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    • 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

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Abstract

The present invention discloses an oximido acetate bactericide detection ELISA method, according to the method, hapten (OESCH2CH2COOH) can be synthesized by use of (E)-2-(2-bromo-methylphenyl)-2-methoxyimino acetate (OEBr), a conjugate is formed by the hapten (OESCH2CH2COOH) and bovine serum albumin (BSA), a healthy rabbit is immunized to obtain a polyclonal antibody, then in-citrus oximido acetate bactericide detection indirect ELISA method is established by use of kresoxim-methyl and trifloxystrobin (oximino acetates bactericides) as standard products and the OESCH2CH2COOH and ovalbumin (OVA) conjugate as a coating antigen, and a method for fast and efficient detection of oximido acetate bactericide residues in citrus is provided, and has the advantages of low cost and good repeatability.

Description

A kind of ELISA method detecting oximide acetic acid ester series bactericidal agent
Technical field
The present invention relates to biological technical field, be specifically related to a kind of the artificial antigen synthesis and the ELISA detection method that detect oximide acetic acid ester series bactericidal agent.
Background technology
Oximide acetic acid ester series bactericidal agent belongs to a class of methoxy acrylic bactericide, and fungicidal spectrum is wide, safe and efficient.Although this kind of toxicity of pesticide is low, because consumption increases year by year, still there is security risk.Therefore both at home and abroad still its residual in food carried out monitoring and formulated strict limit standard, having caused agricultural products in China export's barriers, affect the export economy situation of China.
Current, the instrument detection method such as gas phase, liquid phase that this series bactericidal agent is conventional is highly sensitive, accuracy good, examination scope is wide, and existing comparatively ripe examination criteria, but required expensive equipment, time for sample pretreatment are longer, are unsuitable for field monitoring.
Immunologic detection method (immunoassay, IA) also there is feature that is highly sensitive, high specificity, and numerous and diverse pre-treatment step can be overcome, be widely used in Site Detection, be one of important method of detecting fast of current food security, in food safety detection, play more and more important effect.
Therefore be necessary to develop a kind of immunological technique for detecting the methoxy acrylic bactericide of oximide acetic acid ester class.The present invention mainly chooses kresoxim-methyl and oxime bacterium ester is representative, carries out the foundation of ELISA method.
The poisoning base of oximide acetic acid ester series bactericidal agent is methoxy imino methyl acetate, and (E)-2-(2-2-bromomethylphenyl)-2-methoxyimino methyl acetate (OEBr) is a kind of common pesticide intermediate (Fig. 1, molecular weight < 1000), comprise the active function groups of oximide acetic acid ester series bactericidal agent in structure, therefore it can be used as one of hapten synthesis raw material and mercaptopropionic acid (Fig. 2) to synthesize the artificial semiantigen (AR) of oximide acetic acid ester series bactericidal agent.This haptens is still a kind of small-molecule substance, does not have immunogenicity, after must obtaining artificial antigen, just as animal immune raw material, can prepare polyclonal antibody with couplings such as macromolecular carriers (albumen).
The present invention carries out coupling according to the artificial semiantigen (OESCH2CH2COOH) similar with the chemical constitution of oximide acetic acid ester series bactericidal agent of synthesis and bovine serum albumin and ovalbumin, respectively obtained immunogene and coating antigen.
Summary of the invention
The present invention needs first technical matters solved to be synthetic method and the assay method of the artificial antigen providing a kind of oximide acetic acid ester series bactericidal agent.
1. technical scheme
(1) artificial semiantigen synthesis
In 50mL there-necked flask, add the mercaptopropionic acid ethanolic solution of 5mL1equiv, then add the NaOH of 2equiv, be placed on stirring reaction in the magnetic stirring apparatus of heating function, until dissolve.Add the OEBr ethanolic solution of 1equiv, after heating reflux reaction 1.5h, suction filtration obtains clear solution, rotary evaporation removing organic solvent.Then 5mL5%NaHCO is used 3solubilize residue is also transferred in separating funnel, and extract 2 times with normal hexane, each 5mL, discards organic phase.Then use concentrated hydrochloric acid water transfer phase about pH to 3, and methylene chloride carries out the distribution of liquid liquid, 3 times repeatedly, collect organic phase.Organic phase crosses anhydrous Na 2sO 4, reduced pressure concentration is closely dry, obtains yellow oil.
(2) synthesis of artificial antigen
A. immunogene (active ester method): haptens 0.1mmol and NHS0.1mmol 1.0mLDMF is dissolved.Under magnetic agitation, dropwise add the DMF solution of the 0.1mmolDCC of 1.0mL in above-mentioned solution, stirring at room temperature reaction 1h, stirs in 4 DEG C of refrigerators and spends the night.Next day, centrifuging and taking supernatant, under stirring, supernatant is dropwise joined 6.0mL, in the pH8.0 borate buffer of 60mgBSA, about adds half an hour, room temperature magnetic agitation reaction 1h, 4 DEG C of stirring reactions spend the night, and next day takes out, and solution proceeds in bag filter, be suspended from large beaker, 4 DEG C, the PBS damping fluid of pH7.4 is dialysed 3 days, and every 4h changes water once.Freeze-drying is preserved.
B. coating antigen (mixed anhydride method): take 150mgOVA and be dissolved in the borate buffer of 10mL, the haptens of 80 μm of ol is dissolved in the DMF of 1mL, then in this solution, add tri-n-butylamine and the isobutyl chlorocarbonate of equivalent, room temperature reaction 1h, reactant liquor 500 μ L joins in the OVA borate buffer of the 15mg/mL of 8mL, magnetic agitation, reaction 2h.After having reacted, proceed to bag filter, first use distill water dialysis 1 time, then change the PBS dialysis of pH7.4, whole dialysis procedure continues three days, within every 4 hours, changes dislysate once.Freeze-drying is preserved.
(3) the present invention has carried out open explanation to the authentication method of prepared artificial semiantigen and antigen simultaneously, and method comprises TNBS development process, infra-red sepectrometry, ultraviolet spectroscopy, SDS-PAGE, specifically comprises the following steps:
1) qualification of artificial semiantigen: the haptenic sample taking synthesis, suitably be diluted to suitable detectable concentration, carry out TLC (Fig. 3), ultraviolet (Fig. 4), infared spectrum scanning (Fig. 5), mass spectrum (Fig. 6) and nuclear-magnetism (Fig. 7).Read the position of haptenic crest and trough and analyze the infrared absorption situation of haptenic functional group, the atom of molecular weight and each functional group forms.
2) qualification of artificial immunogen and coating antigen: BSA distilled water is mixed with concentration be 0,0.6,0.7,0.8,0.9, the solution of 1.0mg/mL, get the carbonate that 1mL joins 1mLpH10 and delay colorimetric.Typical curve is made according to light absorption value and protein concentration.The light absorption value of slope and BSA unit concentration.
The haptens distilled water of preparation is mixed with concentration be 0,0.6,0.7,0.8,0.9, the solution of 1.0mg/mL, all the other steps are the same, make curve, and rate of curve is the light absorption value of sample unit concentration.
A. amino consumption rate=(OD protein units concentration-OD sample unit concentration)/OD protein units concentration
B.AR is combined the free amine group major part of the amino number carrier protein of the amino consumption rate × per molecule carrier protein of ratio=AR from lysine with carrier protein grammol, per molecule BSA and OVA lysine number are in 56 and 20.
Meanwhile, the antigen of preparation is carried out ultraviolet (Fig. 4), infrared (Fig. 8), SDS-PAGE (Fig. 9), the concentration of BSA contained by must ensureing in same level, analytical structure and molecular weight.
The object haptens of this method synthesis remains the chemical constitution of oximide acetic acid ester series bactericidal agent poisoning base, and be conducive to the specificity of follow-up immunoassay, building-up process is easy, is applicable to a large amount of production.
Object artificial immunogen and the coating antigen of this method synthesis meet immune requirement, and operation easily realizes, can commercialization.
(4) on this basis, Section 2 technical matters of the present invention is the immunogen immune animal with preparation, obtain the polyclonal antibody for oximide acetic acid ester series bactericidal agent, the carrying out of this antibody is identified and sets up with the ELISA detection method of kresoxim-methyl and the oxime bacterium ester oximide acetic acid ester series bactericidal agent that is representative.
First the polyclonal antibody preparing high-titer and high specific will have desirable immunogene, suitable animal and feasible immunization method.At present, the domestic rare report of immuno analytical method about oximide acetic acid ester series bactericidal agent, there is no the foundation of method for quick.The oximide acetic acid ester series bactericidal agent ELISA detection technique that the present invention sets up, better, expense is low for sensitivity and specificity, and instrumentation requires low, and pre-treatment is easy, has considerable economic benefit.
The concrete technical scheme of polyclonal antibody preparation is as follows:
1) animal used as test: Male New Zealand White Rabbit, body weight is about 2.5kg;
2) immunity and blood sampling: head exempts from first 7 days, and auricular vein gets blood, is separated negative serum.First immunisation, mixes immunogene (2mg/mL) with isopyknic FCA, fully emulsified, forms water in oil state, dorsal sc 6 point, hind leg muscle 2 immunity; Two weeks, interval, secondary immunity, immunogene (1mg/mL) and isopyknic FIA prepare vaccine, dorsal sc 6 point, hind leg muscle 2 immunity; Until the 7th time from third time immunity, immunogene (1mg/mL) and isopyknic FIA prepare vaccine, dorsal sc 6 point, hind leg muscle 2 immunity, and with two weeks for interval, carry out the blood sampling of ear edge, separation of serum, survey is tired; End is exempted from, and now tires and reaches certain level (3.2 × 10 4~ 6.4 × 10 4), substituted by adjuvant physiological saline, auricular vein is injected, after one week, and arterial blood extracting, separation of serum.
The concrete steps that enzyme linked immunosorbent detection is tired are:
1) coating antigen wrapper sheet: Hapten-OVA coating buffer is diluted to debita spissitudo, every hole 100 μ L adds 96 hole ELISA Plate, covers preservative film, hatches 2h for 37 DEG C;
2) cleaning of enzyme target: remove liquid in hole rapidly, wash 2 times with PBST, wash 3min at every turn;
3) close: every hole adds 200 μ L confining liquids, is placed in 37 DEG C of constant incubators and closes 2h; Then pat dry after washing 3 times with PBST;
4) antiserum is added: by 1: 10 3~ 1: 128 × 10 3gradient dilution antiserum, add in enzyme mark reacting hole, every sample at least repeats once, and every hole adds 100 μ L, hatches half an hour for 37 DEG C, and turned letter, washs 3 times, often all over 3 minutes, pats dry.
5) add two to resist: HRP enzyme mark goat anti-rabbit igg is diluted 1000 times, and every hole adds 50 μ L37 DEG C, 30-60min, evacuation, washes plate 3 times, each 3 minutes, pats dry;
6) add TMB chromogenic reagent: every hole 100 μ L, put 37 DEG C of lucifuges and place 10-15min.
7) cessation reaction: every hole adds 100 μ L stop buffers and terminates reaction, should in 20min measurement result.
8) microplate reader detects: after TMB reaction, microplate reader 450nm wavelength reads OD 450.If blank and negative control, the negative rabbit anteserum gathered before being respectively PBS solution and immunity.
(5) Section 3 technical matters of the present invention is the application of setting up a kind of described oximide acetic acid ester series bactericidal agent immunologic detection method, the method is applied to the detection of kresoxim-methyl and oxime bacterium ester in oranges and tangerines, the tire antibody of the highest (64000) of utilization carries out square formation titration, selects OD 450the antigen of ≈ 1, antibody concentration are as best effort concentration, and namely antigen concentration is 10 μ g/mL, antibody dilution 2000 times.
Concrete steps are:
1) coating antigen wrapper sheet: Hapten-OVA coating buffer is diluted to debita spissitudo, every hole 100 μ L adds 96 hole ELISA Plate, covers preservative film, hatches 2h for 37 DEG C;
2) cleaning of enzyme target: remove liquid in hole rapidly, wash 2 times with PBST, wash 3min at every turn;
3) close: every hole adds 200 μ L confining liquids, is placed in 37 DEG C of constant incubators and closes 2h; Then pat dry after washing 3 times with PBST;
4) add antibody: dilute after antibody with volume ratio 1: 2000 with sample diluting liquid, spend the night with standard items (or sample) 1: 1 premix, every hole adds 100 μ L, to hatch after half an hour washing (PBST) 3-4 time for 37 DEG C.
5) add two to resist: HRP enzyme mark goat anti-rabbit igg is diluted 1000 times, and every hole adds 50 μ L37 DEG C, 30-60min, evacuation, washes plate 3 times, each 3 minutes, pats dry;
6) add TMB chromogenic reagent: every hole 100 μ L, put 37 DEG C of lucifuges and place 10-15min.
7) cessation reaction: every hole adds 100 μ L stop buffers and terminates reaction, should in 20min measurement result.
8) microplate reader detects: after TMB reaction, microplate reader 450nm wavelength reads OD 450.If blank and negative control, the negative rabbit anteserum gathered before being respectively PBS solution and immunity.
9) the PBST damping fluid configuration concentration containing 10% methyl alcohol is used to be 200,20,2,0.2,0.02,0.002 μ g/mLHapten, kresoxim-methyl, oxime bacterium ester standard solution.By IC-ELISA method detection sensitivity, calculate concentration IC in the suppression of three 50and method lowest detectable limit IC 10.
10) adopt the azoles bacterium ammonium ester similar to oximide acetic acid ester series bactericidal agent, carry out cross reaction by the detection method set up.With being 200,20 containing the PBST damping fluid configuration concentration of 10% methyl alcohol, the azoles bacterium ammonium ester solution of 2,0.2,0.02,0.002 μ g/mL, by IC-ELISA method detection specificity, calculates IC 50with cross reacting rate CR=[IC 50(Hapten)/IC 50(azoles bacterium ammonium ester)].
11) tangerine meat sample making beating, takes 10.0g, revolves nest mixing, 4 DEG C of standing half an hour, medicine is fully contacted with sample after adding variable concentrations kresoxim-methyl and oxime bacterium ester titer respectively.Rear taking-up, 8000rpm refrigerated centrifuge, gets supernatant.Adopt the IC-ELISA detection method of above-mentioned foundation, measure the recovery and RSD%.
2. benefit result
Above sensitivity and the display of specificity experiments method, haptenic IC 50be 9.5 μ g/mL, IC 10be 0.005 μ g/mL; Antibody has good specificity and sensitivity, IC to kresoxim-methyl and oxime bacterium ester 50be respectively 15.3,19.6 μ g/mL, IC 10be respectively 0.0074,0.011 μ g/mL; But 0.1% is less than to the cross reacting rate of azoles bacterium ammonium ester.The method has good specificity and sensitivity.
Adopt the IC-ELISA set up to detect kresoxim-methyl and oxime bacterium ester in oranges and tangerines sample, testing result shows when spiked levels is 100,10, during 1 μ g/mL, the kresoxim-methyl recovery is respectively 104.35%, and 95.57%, 82.73%, RSD% is respectively 11.1%, 10.8%8.8%; The oxime bacterium ester recovery is respectively 92.43%, 91.75%, 91.10%, RSD% and is respectively 7.4%, 10.2%, and 10.9% the method meets the requirement of germifuge trace analysis.
Embodiment
1. instrument
2. reagent
Bovine serum albumin(BSA) (BSA), oralbumin (OVA) are purchased from sigma company; N-hydroxysuccinimide (NHS), R250 Coomassie brilliant blue, N, N '-dicyclohexylcarbodiimide (DCC), tri-n-butylamine, isobutyl chlorocarbonate, absolute ethyl alcohol, methylene chloride, methyl alcohol, DMF, boric acid, borax, NaOH, sodium bicarbonate is purchased from Hangzhou Mick chemical industry; Tris-base, acrylamide, N, N '-methylene bisacrylamide, Ammonium Persulfate 98.5 (AP), N, N, N ', N '-tetramethylethylenediamine (TEMED), DTT, glycocoll, purchased from Hangzhou Lan Bao biotech firm.Freund's complete adjuvant (FCA) and incomplete Freund's adjuvant (FIA) are purchased from sigma company.GoatAnti-rabbitIgG/HRP is provided by the Shanghai biological company limited of rich credit.TMB one-component nitrite ion, deuterated DMSO is purchased from Aladdin Reagent Company.
The haptenic synthesis of embodiment 1 oximide acetic acid ester series bactericidal agent
Undertaken by Figure 11 by following haptenic synthetic reaction approach.Concrete steps are as follows: in 50mL there-necked flask, add the mercaptopropionic acid ethanolic solution of 5mL1equiv, then add the NaOH of 2equiv, be placed on stirring reaction in the magnetic stirring apparatus of heating function, until dissolve.Add the OEBr ethanolic solution of 1equiv, after heating reflux reaction 1.5h, suction filtration obtains clear solution, rotary evaporation removing organic solvent.Then 5mL5%NaHCO is used 3solubilize residue is also transferred in separating funnel, and extract 2 times with normal hexane, each 5mL, discards organic phase.Then use concentrated hydrochloric acid water transfer phase about pH to 3, and methylene chloride carries out the distribution of liquid liquid, 3 times repeatedly, collect organic phase.Organic phase crosses anhydrous Na 2sO 4, reduced pressure concentration is closely dry, obtains yellow oil.
The synthesis of embodiment 2 oximide acetic acid ester series bactericidal agent artificial antigen
1. the reaction path of immunogene synthesis
Undertaken by the reaction path of Figure 12 immunogene synthesis.Concrete steps are: dissolved by haptens 0.1mmol and NHS0.1mmol 1.0mLDMF.Under magnetic agitation, dropwise add the DMF solution of the 0.1mmolDCC of 1.0mL in above-mentioned solution, stirring at room temperature reaction 1h, stirs in 4 DEG C of refrigerators and spends the night.Next day, centrifuging and taking supernatant, under stirring, supernatant is dropwise joined 6.0mL, in the pH8.0 borate buffer of 60mgBSA, about adds half an hour, room temperature magnetic agitation reaction 1h, 4 DEG C of stirring reactions spend the night, and next day takes out, and solution proceeds in bag filter, be suspended from large beaker, 4 DEG C, the PBS damping fluid of pH7.4 is dialysed 3 days, and every 4h changes water once.Freeze-drying is preserved.
2. the reaction path of coating antigen synthesis
Undertaken by the reaction path of Figure 13 coating antigen synthesis.Concrete steps are: take 150mgOVA and be dissolved in the borate buffer of 10mL, the haptens of 80 μm of ol is dissolved in the DMF of 1mL, then in this solution, add tri-n-butylamine and the isobutyl chlorocarbonate of equivalent, room temperature reaction 1h, reactant liquor 500 μ L joins in the OVA borate buffer of the 15mg/mL of 8mL, magnetic agitation, reaction 2h.After having reacted, proceed to bag filter, first use distill water dialysis 1 time, then change the PBS dialysis of pH7.4, whole dialysis procedure continues three days, within every 4 hours, changes dislysate once.Freeze-drying is preserved.
The mensuration of embodiment 3 antigen
The yellow oil methyl alcohol of acquisition is dissolved, carries out Mass Spectrometer Method; Dissolve with deuterated DMSO and carry out nuclear-magnetism detection; Dissolve with pure water and carry out uv absorption scanning; Infrared spectrum detection is carried out by KBr pressed disc method.
Haptens is at 2000cm -1below have obvious absorption, the scope that the O-H in carboxylic acid stretches is at 1720-1706cm -1, there is the carboxyl characteristic peak of this absorption region in figure, containing COOH-in certification structure; At 1750cm -1place can see that a unconspicuous absorption peak is the absorption peak of C=O, this is because carbonyl and phenyl ring ring conjugation reduce absorption frequency.At 1600 ~ 1450cm -1have obvious one group of absorption peak, deduction is the vibration peak of due phenyl ring skeletal vibration peak and C=N double bond thereof in haptens, at 1250cm -1there is-SCH at place 2-in-CH 2-wagging vibration absorption peak.Prove that compound contains all characteristic absorption peaks of object thus, haptens has no obvious crest and trough at ultraviolet all band place.Mass spectrum nuclear-magnetism surveys molecular weight and structure all meets expected results, the success of preliminary deduction hapten synthesis.
The mensuration of embodiment 4 conjugate
Spectral scan and electrophoresis detection are carried out to coupled product.BSA and OVA has obvious trough at 230-240nm place, and haptens does not then have, simultaneously Hapten-BSA and Hapten-OVA trough herein is obviously covered a part, illustrates that BSA and OVA is still maintain the original character of albumen by hapten transformation.The electrophoretic band of conjugate comparatively albumen relatively lags behind, and illustrates that haptens and albumen have carried out coupling.
Simultaneously test concrete in conjunction with ratio.Adopt 2,4,6-trinitro-benzene-sulfonic acid (TNBS) colourimetry.BSA distilled water is mixed with concentration be 0,0.6,0.7,0.8,0.9, the solution of 1.0mg/mL, get the carbonate that 1mL joins 1mLpH10 and delay colorimetric.Typical curve is made according to light absorption value and protein concentration.The light absorption value of slope and BSA unit concentration.
Sample distilled water is mixed with concentration be 0,0.6,0.7,0.8,0.9, the solution of 1.0mg/mL, all the other steps are the same, make curve, and rate of curve is the light absorption value of sample unit concentration.
A. amino consumption rate=(OD protein units concentration-OD sample unit concentration)/OD protein units concentration
B.AR is combined the free amine group major part of the amino number carrier protein of the amino consumption rate × per molecule carrier protein of ratio=AR from lysine with carrier protein grammol, per molecule BSA and OVA lysine number are in 56 and 20.
Obtain combining than bioassay standard curve by the described method in conjunction with ratio: (BSA) y=1.646x-0.1542, R 2=0.9976.(OVA)y＝1.712x-0.1431R 2＝0.9916。Therefore, the light absorption value of BSA and OVA unit concentration is respectively 1.646, and 1.712.Immunogene sample determination combines than curve: y=1.269x-0.3082, R 2=0.9568.Therefore, the light absorption value of sample unit concentration is 1.269.As calculated: [(1.646-1.269)/1.646] × 56 ≈ 12.8.Too much haptens may be unfavorable for that carrier is combined with lymphocytic cell surface, and carrier can not be made to cause immune response.For BSA, each molecule connects 5 ~ 20 haptens and be advisable.Therefore this experiment combines than being 12.8 within scope, and coupling effect is good.And as coating antigen, it combines is y=0.954x-0.2132 than curve, R 2=0.9778.Therefore, the light absorption value of sample unit concentration is 1.344.As calculated: [(1.712-0.954)/1.712] × 20 ≈ 8.8.
Determine thus, haptens and the success of BSA and OVA even-coupling, and meet the desired condition of immuno analytical method.
The preparation of embodiment 5 polyclonal antibody
Adopting the immunogene of Prof. Du Yucang and Freundadjuvant is fully emulsified makes vaccine, immunize New Zealand White Rabbit, tiring by measuring, the more much higher clonal antibody of acquisition susceptibility.
1. immunologic process
Head exempts from first 7 days, and auricular vein gets blood, is separated negative serum.First immunisation, mixes immunogene (2mg/mL) with isopyknic FCA, fully emulsified, forms water in oil state, dorsal sc 6 point, hind leg muscle 2 immunity; Two weeks, interval, secondary immunity, immunogene (1mg/mL) and isopyknic FIA prepare vaccine, dorsal sc 6 point, hind leg muscle 2 immunity; Until the 7th time from third time immunity, immunogene (1mg/mL) and isopyknic FIA prepare vaccine, dorsal sc 6 point, hind leg muscle 2 immunity, and with two weeks for interval, carry out the blood sampling of ear edge, separation of serum, survey is tired; End is exempted from, and now tires and reaches certain level (3.2 × 10 4~ 6.4 × 10 4), substituted by adjuvant physiological saline, auricular vein is injected, after one week, and arterial blood extracting, separation of serum.
2. the compound method of reagent needed for
As Figure 14
3.ELISA reaction principle
1) add antigen: coating antigen coating buffer is diluted to 10 μ g/mL, every hole 100 μ L adds 96 hole ELISA Plate, covers preservative film 37 DEG C and hatches 2h, closes washing according to a conventional method;
2) add antibody: dilute after antibody with volume ratio 1: 2000 with sample diluting liquid, spend the night with standard items (or sample) 1: 1 premix, every hole adds 100 μ L, to hatch after half an hour washing (PBST) 3-4 time for 37 DEG C.
3) add two to resist: HRP enzyme mark goat anti-rabbit igg is diluted 1000 times, and every hole adds 50 μ L37 DEG C, 30-60min, evacuation, washes plate 3 times, each 3 minutes, pats dry;
4) add TMB chromogenic reagent: every hole 100 μ L, put 37 DEG C of lucifuges and place 10-15min;
5) cessation reaction: every hole adds 100 μ L stop buffers and terminates reaction, should in 20min measurement result;
6) microplate reader detects: after TMB reaction, microplate reader 450nm wavelength reads OD 450; If blank and negative control, the negative rabbit anteserum gathered before being respectively PBS solution and immunity.
4.IC-ELISA method specific detection
Specific detection is for measuring the reaction capacity of kresoxim-methyl, oxime bacterium ester and antibody.Adopt the antigen of best effort concentration and the condition of antibody.Adopt the azoles bacterium ammonium ester being all Strobilurin series bactericidal agent, carry out cross reaction by the detection method set up.With being 200,20 containing the PBST damping fluid configuration concentration of 10% methyl alcohol, the azoles bacterium ammonium ester solution of 2,0.2,0.02,0.002 μ g/mL, by IC-ELISA method detection specificity, calculates IC 50with cross reacting rate CR=[IC 50(Hapten)/IC 50(azoles bacterium ammonium ester)].
5.IC-ELISA method sensitivity detects
With being 200,20 containing the PBST damping fluid configuration concentration of 10% methyl alcohol, 2,0.2,0.02,0.002 μ g/mLHapten, kresoxim-methyl, oxime bacterium ester standard solution.By IC-ELISA method detection sensitivity, calculate concentration IC in the suppression of three 50and method lowest detectable limit IC 10, result is as Figure 15.
The cross reacting rate CR=IC of azoles bacterium ammonium ester (Pyraclostrobin) 50(Hapten)/IC 50(Pyraclostrobin) < 0.1%.Because not containing methoxyimino methyl acetate in its structure, react so do not compete with antibody after adding series concentration.Polyclonal antibody specificity is better, has good affinity to oximide acetic acid ester class (kresoxim-methyl, the oxime bacterium ester etc.) germifuge containing haptens structure, can not effectively identify the germifuge not containing this structure.
6. sample detection and the recovery
Tangerine meat sample is pulled an oar, and takes 10.0g, revolves nest mixing, 4 DEG C of standing half an hour, medicine is fully contacted with sample after adding variable concentrations kresoxim-methyl and oxime bacterium ester titer respectively.Rear taking-up, 8000rpm refrigerated centrifuge, gets supernatant.Adopt the IC-ELISA detection method of above-mentioned foundation, measure the recovery and RSD%.The results are shown in Figure 16, substantially meet trace detection requirement.
Figure of description
Fig. 1 is hapten synthesis raw material OEBr
Fig. 2 is hapten synthesis raw material 2 mercaptopropionic acid
Fig. 3 is hapten synthesis TLC result 1. mercaptopropionic acid 2.OEBr3. reactant liquor
Fig. 4 is artificial antigen ultraviolet all band scintigram
Fig. 5 is haptens infrared scan figure
Fig. 6 is haptens mass spectrum
Fig. 7 is haptens nucleus magnetic hydrogen spectrum
Fig. 8 be haptens-BSA (on) and haptens-OVA (under) infrared scan figure
Fig. 9 is the protein electrophoresis figure of artificial antigen
Figure 10 is kresoxim-methyl (left side) and oxime bacterium ester (right side)
The reaction path of Figure 11 hapten synthesis
The reaction path of Figure 12 immunogene synthesis
The reaction path of Figure 13 coating antigen synthesis
The compound method of reagent needed for Figure 14
Figure 15 antibody is to the cross reaction of not synantigen
Figure 16 recovery of standard addition and precision result

Claims (4)

1. detect an ELISA method for oximide acetic acid ester series bactericidal agent, comprise following content:
(1) be Material synthesis artificial antigen by reactive group oximide acetic acid ester ((E)-2 (2-2-bromomethylphenyl)-2-methoxyimino methyl acetate (OEBr));
(2) preparation containing oximide acetic acid ester series bactericidal agent artificial antigen vaccine and immunize New Zealand White Rabbit prepares polyclonal antibody;
(3) set up the indirect ELISA detection method detecting oximide acetic acid ester series bactericidal agent, measure the residual of germifuge in different sample.
2. the ELISA detection method of a kind of oximide acetic acid ester series bactericidal agent according to claim 1, it is characterized in that, in the synthesis of oximide acetic acid ester series bactericidal agent artificial semiantigen, the rate of charge of mercaptopropionic acid ethanolic solution, NaOH, OEBr ethanolic solution is 1: 2: 1equiv; Adopt methylene chloride to carry out pH when liquid liquid distributes and should be about 2.8-3.2.
3. the ELISA detection method of a kind of oximide acetic acid ester series bactericidal agent according to claim 1, it is characterized in that, in oximide acetic acid ester series bactericidal agent artificial antigen (immunogene and coating antigen) preparation, haptens and the mol ratio of NHS of synthesis are 1: 1, for dropwise to add when adding DMF solution; The rate of charge of tri-n-butylamine and isobutyl chlorocarbonate is 1: 1equiv; The ratio of quality and the number of copies of described haptens and BSA or OVA is 60 ~ 150: 1; The pH preparing immunogenic whole system should be 7.4, and the whole system preparing coating antigen is 8.0; Utilize immunogene to mix with immunologic adjuvant, preparation containing the vaccine of oximide acetic acid ester series bactericidal agent artificial antigen and immunize New Zealand White Rabbit, takes White Rabbit blood and separation of serum, preparation can with the polyclonal antibody of oximide acetic acid ester series bactericidal agent idiosyncrasy.
4. the ELISA detection method of a kind of oximide acetic acid ester series bactericidal agent according to claim 1, it is characterized in that, in enzyme linked immunosorbent detection, coating antigen coating buffer is diluted to 10 μ g/m; After diluting antibody with sample diluting liquid with volume ratio 1: 2000, spend the night with standard items (or sample) 1: 1 premix; HRP enzyme mark goat anti-rabbit igg is diluted 1000 times.
CN201410200830.9A 2014-05-12 2014-05-12 Oximido acetate bactericide detection ELISA method Pending CN105092833A (en)

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