CN107188830B - A kind of pyrethrin pesticide haptens and its preparation method and application - Google Patents

A kind of pyrethrin pesticide haptens and its preparation method and application Download PDF

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CN107188830B
CN107188830B CN201710466703.7A CN201710466703A CN107188830B CN 107188830 B CN107188830 B CN 107188830B CN 201710466703 A CN201710466703 A CN 201710466703A CN 107188830 B CN107188830 B CN 107188830B
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haptens
pyrethrin pesticide
monoclonal antibody
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pyrethrin
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CN107188830A (en
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万宇平
吴小胜
崔海峰
宋灏
贾芳芳
何方洋
顾蓉蓉
刘玉梅
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Beijing Kwinbon Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/38—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by esterified hydroxy groups
    • C07C255/39—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by esterified hydroxy groups with hydroxy groups esterified by derivatives of 2,2-dimethylcyclopropane carboxylic acids, e.g. of chrysanthemumic acids
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • 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

Abstract

The present invention relates to a kind of haptens and its preparation method and application, and in particular to a kind of pyrethrin pesticide haptens and its preparation method and application.Based on the pyrethroids class monoclonal antibody hybridoma cell strain F-3-2 of pyrethrin pesticide haptens screening, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.13842.The pyrethroids class antibody titer of this cell strain secretion reaches 2 × 105Cross reacting rate with decis is 100%, cross reacting rate with lambda-cyhalothrin is 50.9%, cross reacting rate with taufluvalinate is 103.6%, cross reacting rate with cyfloxylate is 70.7%, and the cross reacting rate with penta polyester of chlorine is 74.4%, and the cross reacting rate with benzene Permethrin is 69.0%, condition is provided for the how remaining immune detection of pyrethrin pesticide, there is practical application value.

Description

A kind of pyrethrin pesticide haptens and its preparation method and application
Technical field
The present invention relates to a kind of haptens and its preparation method and application, and in particular to pyrethrin pesticide haptens and its system Preparation Method and application belong to food safety technical field of immunological detection.
Background technique
Pyrethrin pesticide have the characteristics that insecticidal spectrum is wide, drug effect rapidly, it is long to light and thermally stable, efficacy time, agricultural, The fields such as forestry, public health are widely used, but it has harm to the ecosystem and human health, especially to honeybee and aquatic Biology has high toxicity.The World Health Organization (WHO) and FAO (Food and Agriculture Organization of the United Nation) (FAO) are residual on vegetables and fruits to pyrethrin pesticide It stays and has formulated stringent limit standard, China has also formulated limit standard (GB 2763-2016) to such pesticide.
Currently, the detection method of pyrethrin pesticide is mainly chromatography, including gas-chromatography (GC), high performance liquid chromatography (HPLC), mass spectrography (MS) and gas chromatography-mass spectrography (GC-MS) or Liquid Chromatography-Mass Spectrometry (HPLC-MS) etc.. Instrument analytical method has the advantages that high sensitivity, but expense is costly, and technical requirements are high, and it is a large amount of to be not suitable for on-site quick screening Sample.
Immunoassay can make up all of above disadvantage, immunoassay be with specific recognition between antigen and antibody and Trace analysis methods based on invertibity association reaction, since with high specificity, high sensitivity, simple, quick, expense is low It the advantages that with live batch samples screening is suitable for, is used widely in the analysis fields such as environment and food safety.It builds The key of the immunoassay method of vertical small molecule compound is can to prepare to have high-affinity and height to small molecule compound Antibody selective, and the synthesis of haptens is the basis for preparing high-quality antibody.The key of hapten design is as much as possible Retain the feature structure of former test substance, and introduces suitable linking arm and the activity with carrier protein couplet in position Group.The introducing position of the linking arm of different structure, different coupling methods or linking arm, all influences the antibody of subsequent preparation Sensitivity and specificity.In addition, as the same clan, family compound, a variety of pyrethrin pesticides are in food and the cumulative toxicity in environment There is wide spectrum to know for serious implicit risk, therefore synthesizing efficient valence, the general antigen with pyrethroids class structural features, preparation Other antibody becomes the emphasis of pyrethrin pesticide immunoassay research.
Summary of the invention
The purpose of the present invention is to provide a kind of pyrethrin pesticide haptens and preparation method thereof and the haptens and people The monoclonal antibody that the conjugate that seralbumin is coupled is prepared as immunogen immune animal.It is verified through test data, The monoclonal antibody of preparation is to decis, lambda-cyhalothrin, taufluvalinate, cyfloxylate, chlorine valerate, benzene Permethrin 6 kinds of pyrethrin pesticides have preferable detection sensitivity, can be used to establish the how remaining immunology detection side of pyrethrin pesticide Method.
The purpose of the present invention is achieved by the following technical programs:
A kind of pyrethrin pesticide haptens, molecular structural formula are as follows:
The preparation method of above-mentioned pyrethrin pesticide haptens, includes the following steps:
[cyano-(3- phenoxy group)-methyl] 2 bromacetate 1.0g are taken, adds n,N-Dimethylformamide to dissolve, adds bicarbonate Sodium 0.72g after mixing is sufficiently stirred, adds 1- amino -2,2- dimethyl cyclopropane carboxylic acid 0.45g, 60 DEG C are stirred to react 4h；Stop Reaction adds water ethyl acetate to extract, and stratification, organic phase anhydrous sodium sulfate is dry, is evaporated, upper silicagel column, petroleum ether/acetic acid Ethyl ester=1:1 elution separation, obtains cyclopropane carboxyl pyrethroids haptens 1.1g, as pyrethrin pesticide haptens, yield 97.35%.
Application of the above-mentioned pyrethrin pesticide haptens in immune detection, specifically includes by the pyrethrin pesticide haptens The pyrethrin pesticide artificial antigen obtained with carrier protein couplet, and animal system is immunized by resulting pyrethrin pesticide artificial antigen Standby monoclonal antibody.
Wherein the carrier protein be mouse serum albumin, thyroprotein, bovine serum albumin(BSA), albumin rabbit serum, Human serum albumins, ovalbumin, hemocyanin or fibrinogen.
The monoclonal antibody is thin for the pyrethroids class monoclonal antibody hybridoma of CGMCC No.13842 by deposit number Born of the same parents' strain F-3-2 secretion generates, more residual immune detections applied to pyrethrin pesticide.
The beneficial effects of the present invention are:
(1) pyrethrin pesticide haptens provided by the invention had both farthest remained the chemistry of pyrethrin pesticide parent nucleus Structure, further through chemical synthesis transformation introduce can with protein be coupled-COOH, synthetic method is simple, purity, yield It is higher；
(2) monoclonal antibody that animal obtains is immunized in the pyrethrin pesticide artificial antigen that the present invention is prepared, and potency reaches To 2 × 105, illustrate that the antigen that the present invention synthesizes has excellent immunogenicity, completely remained during synthetic antigen The prototype structure of hapten molecule；
(3) monoclonal antibody specificity provided by the invention is good, and the cross reacting rate with decis is 100%, with chlorine The cross reacting rate of flucythrinate is 50.9%, and the cross reacting rate with taufluvalinate is 103.6%, with cyfloxylate Cross reacting rate is 70.7%, and the cross reacting rate with penta polyester of chlorine is 74.4%, and the cross reacting rate with benzene Permethrin is 69.0%, it can be applied to more residual immune detections of pyrethrin pesticide.
Biological material specimens preservation: one plant of pyrethroids class monoclonal antibody hybridoma cell strain has been preserved in China Microbiological Culture presevation administration committee common micro-organisms center, abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, preservation date on May 05th, 2017, deposit number was CGMCC No.13842.
Detailed description of the invention
Fig. 1 pyrethrin pesticide hapten synthesis route map
Fig. 2 pyrethrin pesticide haptens hydrogen nuclear magnetic resonance spectrogram
Fig. 3 pyrethrin pesticide competitive ELISA canonical plotting
Specific embodiment
Present invention be described in more detail in the following with reference to the drawings and specific embodiments, it should be appreciated that these embodiments are only used for Illustrate the present invention, and is not intended to limit the scope of the invention.Experimental method used in following embodiments unless otherwise specified, It is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1: pyrethrin pesticide hapten synthesis and identification
The synthetic route of pyrethrin pesticide haptens is as shown in Fig. 1.
[cyano-(3- phenoxy group)-methyl] 2 bromacetate 1.0g are taken, adds n,N-Dimethylformamide to dissolve, adds bicarbonate Sodium 0.72g after mixing is sufficiently stirred, adds 1- amino -2,2- dimethyl cyclopropane carboxylic acid 0.45g, 60 DEG C are stirred to react 4h；Stop Reaction adds water ethyl acetate to extract, and stratification, organic phase anhydrous sodium sulfate is dry, is evaporated, upper silicagel column, petroleum ether/acetic acid Ethyl ester=1:1 elution separation, obtains cyclopropane carboxyl pyrethroids haptens 1.1g, as pyrethrin pesticide haptens, yield 97.35%.
Above-mentioned haptens is taken to identify that the results are shown in attached figure 2 through nuclear magnetic resonance spectroscopy.1H-NMR(CDCl3, 300MHz) and δ: 11.00 (1H, s, COOH), 7.41 (2H, dd, ArH), 7.17 (1H, dd, ArH), 7.14 (2H, dd, ArH), 7.08 (2H, dd, ArH), 7.34 (2H, dd, ArH), 6.25 (1H, s, CH), 3.51 (2H, s, CH2), 2.0 (1H, s, NH), 0.94 (6H, s, CH3), 0.57 (1H, d, CH), 0.33 (1H, d, CH).In map, chemical shift δ=11.0 are carboxyl hydrogen resonance absorbing peak on compound, 7.08~7.41 be the absorption peak of hydrogen on phenyl ring, and 0.94 is the absorption peak of two methyl hydrogens, and 0.33~0.57 is on three-membered ring Two hydrogen absorption peaks, in conjunction with other absorption peaks, it was demonstrated that hapten synthesis success.
Embodiment 2: the synthesis of pyrethrin pesticide artificial antigen and identification
It is even with mixed anhydride method and human serum albumins (HSA) using the carboxyl of pyrethrin pesticide haptens as active site Connection prepares immunogene；It is coupled with carbodlimide method and ovalbumin (OVA), prepares coating antigen.
1. mixed anhydride method prepares immunogene
Cyclopropane carboxyl pyrethroids haptens 24mg is taken, dimethyl sulfoxide 0.5mL is added to dissolve, adds 20 μ L of triethylamine, stirring is mixed Even, 20 μ L of chlorination iso-butyl formate stirs 30min, obtains haptens activating solution A liquid；HSA 100mg is taken, adds 0.1mol/L's Phosphate buffer dissolution, obtains B liquid；A liquid is added drop-wise to dropwise in B liquid, 4h is stirred at room temperature, the phosphate of 0.01mol/L is slow Fliud flushing is dialysed 3 days, is changed liquid 3 times daily, is obtained immunogene, -20 DEG C save backup.
2. carbodlimide method prepares coating antigen
Cyclopropane carboxyl pyrethroids haptens 10mg is taken, n,N-Dimethylformamide (DMF) 0.3mL is added to dissolve, adds carbon two sub- Amine (EDC) 13mg stirs 30min, adds n-hydroxysuccinimide (NHS) 13mg, stirs 30min, obtains haptens activating solution A Liquid；OVA 100mg is taken, adds the phosphate buffer of 0.1mol/L to dissolve, obtains B liquid；A liquid is added drop-wise to dropwise in B liquid, room temperature 4h is stirred, the phosphate buffer of 0.01mol/L dialyses 3 days, changes liquid 3 times daily, obtain coating antigen, -20 DEG C save backup.
Mouse serum albumin, thyroprotein, bovine serum albumin(BSA), the white egg of rabbit anteserum can also be used in the above method White, hemocyanin or fibrinogen.
3. artificial antigen is identified
By carrier protein, pyrethrin pesticide haptens, pyrethrin pesticide hapten-carrier protein conjugate with pH's 7.4 PBS is made into the solution of 0.5mg/mL, is returned to zero with the PBS of 0.01mol/L pH7.4, with ultraviolet specrophotometer wavelength 200~ It is scanned within the scope of 800nm, obtains carrier protein, pyrethrin pesticide haptens, pyrethrin pesticide hapten-carrier protein conjugate Absorption curve, and calculate its combine than.As a result, it has been found that different absorption curves occurs in three, show that pyrethrin pesticide half is anti- Former successful with carrier protein couplet, the combination ratio of haptens and HSA are (17~21): 1, and the combination ratio of OVA is (15~20): 1。
Embodiment 3: preparation and purification of monoclonal antibody and CHARACTERISTICS IDENTIFICATION
1. animal immune
Take 6~8 weeks female Balb/c mouse 10 (be divided into A and two groups of B, every group 5) of health, initial immunity Freund Freund's complete adjuvant emulsifies posterior neck dorsal sc multi-point injection, and every mouse immune dosage is 200 μ g immunogenes；Booster immunization is every later The subcutaneous multi-point injection of the nape of the neck is primary within two weeks, and incomplete Freund's adjuvant is used in emulsification；Last time is immune to be replaced using physiological saline Incomplete Freund's adjuvant, using intraperitoneal injection, injection dosage and front are identical several times.Specifically immune step is shown in Table 1.
1 mouse immune program of table
For the third time, four times, 7d after booster immunization take blood to mouse docking, and ELISA method measures mice serum potency, tool Steps are as follows for body:
(1) coating antigen is done into 1:1000 dilution, every 100 μ L of hole coating with the carbonate buffer solution of 0.05mol/L pH9.6 ELISA Plate, 37 DEG C of incubation 2h, gets rid of coating buffer, with PBST washing 1 time, pats dry；
(2) 150 μ L confining liquids are added in every hole, and 37 DEG C of reaction 2h hypsokinesis deblocking liquid pat dry；
(3) 50 μ L are added with the antiserum of PBS doubling dilution, 25 DEG C of reaction 30min hypsokinesis dereaction liquid, with PBST in every hole Washing 3~5 times, every minor tick 30s is patted dry；
(4) plus 100 hole μ L/ of the diluted ELIAS secondary antibody of PBS (1:1000), 25 DEG C of reaction 30min wash 3~5 with PBST Secondary, every minor tick 30s is patted dry；
(5) substrate developing solution A liquid and each 50 μ L of B liquid is added in every hole, and 25 DEG C are protected from light 15min, and 50 μ are added in every hole The H of L2mol/L2SO4Solution terminates reaction；
(6) microplate reader measures wavelength in the OD value of 450nm, with sample well OD450Extension rate close to 1 is used as the positive The potency of serum.
2. cell fusion
(1) prepared by feeder cells: disconnected neck puts to death 8~10 week old Balb/c mouse, is immersed in 5min in 75% alcohol, immediately It is put into superclean bench, abdomen is put in plate upward or is fixed in dissection plate.Mouse web portion skin is played with ophthalmic tweezers sub-folder Skin cuts an osculum with scissors, pays attention to being sure not to break peritonaeum, in order to avoid peritoneal fluid outflow and pollution.Then use scissors to upper and lower two sides Blunt separation is done, peritonaeum is sufficiently exposed.With cotton ball soaked in alcohol wiping peritonaeum disinfection.The basis 5mL RPMI-1640 is drawn with syringe Culture solution injects mouse peritoneal, gently draws back syringe, shakes mouse leg and tail portion several times.Abdominal cavity is drawn back with original annotation emitter Interior liquid injects centrifuge tube.It operates 3~4 times repeatedly.1000r/min is centrifuged 10min, abandons supernatant.It is complete with 20~50mL Cell is resuspended in culture solution, and 100 holes μ L/ are added drop-wise to culture plate, it is spare to set incubator.
(2) prepared by splenocyte: 3d after booster immunization, takes immune Balb/c mouse, dislocates and put to death after eye socket blood sampling, 75% Spleen is taken after sterilizing in alcohol, is removed connective tissue, is prepared splenocyte suspension, be transferred in 50mL centrifuge tube, add RPMI-1640 It is centrifuged 5min to 30mL, 1500~2000r/min, supernatant is abandoned, adds RPMI-1640 to 30mL, is counted stand-by.
(3) myeloma cell prepare: take good (viable count > 95%) myeloma cell of 3 bottles of growth conditions, by it is complete It full blows down, is transferred in 50mL centrifuge tube, add RPMI-1640 to 30mL, 1500~2000r/min to be centrifuged 5min, abandon supernatant, add RPMI-1640 to 30mL is counted stand-by.
(4) mixing with cells: splenocyte: myeloma cell=8:1, mixing, 1500~2000r/min are centrifuged 5min.
(5) cell fusion: the cell mixed is centrifuged, and dry supernatant sets 37 DEG C of water sedimentation cell block bullet at paste 1mL fusion agent is added in bath in 1min, and fusion agent is polyethylene glycol (PEG) 4000, acts on 2min, and be gently mixed cell, The PEG nutrient solution of 20mL serum-free is added in subsequent 4min, 1000r/min is centrifuged 10min, abandons supernatant.It is complete with 20~50mL Cell is resuspended in culture solution, and cover plant is set in incubator in 96 porocyte culture plates containing feeder cells, every 100 μ L of hole.
3. cell strain screens
When the 1/2~1/3 of cell length to bottom hole, antibody test can be carried out.It is thin to there is hybridoma using ELISA method The culture hole of intracellular growth is screened, and screen in two steps: the first step first filters out positive cell hole, second step with indirect ELISA Selecting a variety of pyrethrin pesticides is standard items, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.It selects to more Kind of pyrethrin pesticide standard items all have the hole preferably inhibited, are subcloned using limiting dilution assay, with same method into Row detection.In triplicate, the cell strain F-3-2 of energy stably excreting pyrethroids class monoclonal antibody can be obtained.The cell strain in It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, ground on May 05th, 2017 Location: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC No.13842。
4. prepared by ascites
Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only.The hybridization of logarithmic growth phase will be in after 10 days Oncocyte CGMCC No.13842 is collected with RPMI-1640 basal medium, and with blood counting chamber and microscopic counting, cell is dense Degree is 1.0 × 106~1.5 × 106Within the scope of a/mL.Every mouse 0.5mL hybridoma CGMCC No.13842 is injected into Abdominal cavity.Notice that observation is expanded in mouse web portion after a week, ascites is acquired in mouse peritoneal with asepsis injector, every one to two days Acquisition is primary, is repeatedly acquired repeatedly until mouse natural death in this way.5000r/min is centrifuged 5min at 4 DEG C, collects supernatant, and Remove the fat and albuminous membranae of the floating of ascites upper layer.
5. antibody purification
Monoclonal antibody is using octanoic acid-ammonium sulfate method purifying, the specific steps are as follows:
(1) ascites is taken out into thaw at RT from -20 DEG C of refrigerators.Ascites is filtered with double-layer filter paper, the preliminary fatty piece, thin of removing Born of the same parents' fragment and other impurities.12000r/min is centrifuged 15min, takes supernatant, abandons precipitating.Precise volume ascites volume；
The ascites of (2) 1 parts of volumes and the acetate buffer magnetic agitation of 3 parts of volumes mix, with 2mol/L HCl tune pH To 4.5~4.8；
(3) caprylic acid is slowly added under magnetic agitation, 1mL ascites adds 33 μ L caprylic acids, adds rear room temperature magnetic agitation 30min, 4 DEG C of standing 2h of postposition；
(4) 12000r/min is centrifuged 5min, takes supernatant, and filtrate is collected in double-layer filter paper filtering；
(5) filtrate volume is measured, the PBS of the 0.1mol/L pH7.4 of 1/10 volume is added, with 2mol/L NaOH (record NaOH volume) adjust pH to 7.4；
(6) supernatant ice bath is pre-chilled, adds ammonium sulfate solids to 0.277g/mL, it is stirring while adding, and in being added in 30min, Set 4 DEG C overnight；
(7) 12000r/min is centrifuged 15min, abandons supernatant.Precipitating is dissolved with the 0.01mol/L PBS of certain volume.Use PB Dialysis changes 0.01mol/L PBS two days later and dialyses two days, collects dialyzate, and 12000r/min is centrifuged 15min, takes supernatant, set -20 DEG C save.
6. antibody titer measures
Use the potency of indirect ELISA method measurement antibody for 1:200000.Specific steps refer to above-mentioned 1. animal immune In content.
7. antibody cross reaction measures
It is measured using indirect competitive ELISA method, specific steps are as follows:
(1) coating antigen is done into 1:1000 dilution, every 100 μ L of hole coating with the carbonate buffer solution of 0.05mol/L pH9.6 ELISA Plate, 37 DEG C of incubation 2h, gets rid of coating buffer, with PBST washing 1 time, pats dry；
(2) 150 μ L confining liquids are added in every hole, and 37 DEG C of reaction 2h hypsokinesis deblocking liquid pat dry；
(3) every hole be first added pyrethrin pesticide standard working solution that 50 μ L are serially diluted (concentration is respectively 0,1,3,9,27, 81 μ g/L), 50 μ L are added then with PBS and do the diluted monoclonal antibody of 1:200000,25 DEG C of reaction 30min hypsokinesis dereactions Liquid, with PBST washing 3~5 times, every minor tick 30s is patted dry；
(4) plus 100 hole μ L/ of the diluted ELIAS secondary antibody of PBS (1:1000), 25 DEG C of reaction 30min wash 3~5 with PBST Secondary, every minor tick 30s is patted dry；
(5) substrate developing solution A liquid and each 50 μ L of B liquid is added in every hole, and 25 DEG C are protected from light 15min, and 50 μ are added in every hole The H of L2mol/L2SO4Solution terminates reaction；
(6) OD value of the microplate reader measurement wavelength in 450nm.
Using the logarithm of pyrethrin pesticide concentration as abscissa, with percentage absorbance value (each concentration standards OD value with not The percentage of the quasi- sample wells OD value of mark-on) it is that ordinate draws standard curve, see attached drawing 3.With each 50% percentage absorbance value of curve Mass concentration (IC50) cross reacting rate is calculated as follows, it the results are shown in Table 2, cross reacting rate is smaller, and antibody specificity is higher.
In formula:
Si--- cross reacting rate, %；
Y --- the IC of decis standard working solution curve50Value；
Z --- the IC of other pyrethrin pesticide standard working solution curves50Value.
2 cross reaction measurement result of table
Note: "-" indicates curve torsional deformation, is not used to calculate.
As shown in Table 2, pyrethrin pesticide monoclonal antibody is to decis, lambda-cyhalothrin, taufluvalinate, cyfluthrin Pyrethroids, penta polyester of chlorine, benzene Permethrin have cross reaction, illustrate that the antibody can be used for the immune inspection of more residuals of pyrethrin pesticide It surveys.

Claims (4)

1. a kind of pyrethrin pesticide artificial antigen, it is characterised in that be to be obtained by pyrethrin pesticide haptens with carrier protein couplet 's；Wherein, the preparation method of the pyrethrin pesticide haptens includes the following steps: to take [cyano-(3- phenoxy group)-methyl]- 2- bromacetate 1.0g, adds n,N-Dimethylformamide to dissolve, and adds sodium bicarbonate 0.72g, after mixing is sufficiently stirred, adds 1- ammonia Base -2,2- dimethyl cyclopropane carboxylic acid 0.45g, 60 DEG C are stirred to react 4h；Stop reaction, adds water-ethyl acetate extraction, stand and divide Layer, organic phase anhydrous sodium sulfate is dry, is evaporated, upper silicagel column, and petrol ether/ethyl acetate=1:1 elution separation obtains cyclopropane Carboxyl pyrethroids haptens, as pyrethrin pesticide haptens, molecular structural formula are as follows:
2. the monoclonal antibody of animal preparation is immunized by pyrethrin pesticide artificial antigen described in claim 1.
3. monoclonal antibody as claimed in claim 2, it is characterised in that it is CGMCC No.13842 that it, which is by deposit number, Pyrethroids class monoclonal antibody hybridoma cell strain F-3-2 secretion generates.
4. monoclonal antibody as claimed in claim 3 is remaining the application in immune detection more pyrethrin pesticide.
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CN106543163A (en) * 2016-11-08 2017-03-29 佛山市飞时达新材料科技有限公司 A kind of haptenic preparation method and applications of fluoxastrobin
CN108535474A (en) * 2018-03-16 2018-09-14 北方工业大学 Time-resolved fluorescence test strip for detecting permethrin and application thereof
CN108931646B (en) * 2018-05-30 2021-04-27 中国烟草总公司郑州烟草研究院 Test strip for detecting safrole and preparation method and application thereof
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