CN103529198B - A kind of test kit for detecting decis and application thereof - Google Patents
A kind of test kit for detecting decis and application thereof Download PDFInfo
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- SPVKRXSQXKGOKK-UHFFFAOYSA-N CC(C)(C1C=C(Br)Br)C1C(NCCCCCC(O)=O)=O Chemical compound CC(C)(C1C=C(Br)Br)C1C(NCCCCCC(O)=O)=O SPVKRXSQXKGOKK-UHFFFAOYSA-N 0.000 description 2
- 0 CC(C)(C1C=C(Br)Br)C1C([*+])=O Chemical compound CC(C)(C1C=C(Br)Br)C1C([*+])=O 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N ON(C(CC1)=O)C1=O Chemical compound ON(C(CC1)=O)C1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
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- 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
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- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
- G01N2430/12—Pyrethroids
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Abstract
The invention discloses a kind of test kit for detecting pesticide deltamethrin, described test kit is by reagent set and is coated plate and forms, described reagent set includes concentrating PBS, chromogenic substrate, concentrated cleaning solution, stop buffer, decis standard solution, decis enzyme-labelled antigen, decis hapten and the conjugate of horseradish peroxidase, dilute 100,000 times with PBS during use.Instant invention overcomes traditional physico-chemical analysis method very complicated, relatively costly, analyze slow problem, it is provided that succinct, quickly, sensitive, immuno analytical method accurately.
Description
Technical field
The invention belongs to the reagent of a kind of detection by quantitative pesticide deltamethrin content in immunological assay techniques field
Box and using method thereof, relate to the technology such as organic chemical synthesis, immunochemistry, biochemistry, especially suitable
The quick detection of deltamethrin residues in vegetable, Folium Camelliae sinensis.
Background technology
Pyrethroid (Pyrethroids) is the biomimetic insecticide that a class is important, is that country advocates popularization
Pesticidal products, according to scholarly forecast, bionic pesticide will be the focus of 21 century novel pesticide exploitation.China is from 20
Century 70 begins to develop this insecticides, its synthesis and application be continue organochlorine insecticide,
A new breakthrough after organophosphorus insecticide, carbamate pesticide, is also the 3rd in insecticide history
Important milestone.It is the agricultural and hygienic insecticide that currently used area is wide, consumption is big, accounts for insecticidal total
The 20% of yield, accounts for the 25% of whole insecticide usable floor area.Along with being continuously increased of its usage amount and kind,
The resistance of this kind of pesticide and residue problem have caused the extensive concern of people.To this end, residual to pesticide deltamethrin
Stay restriction more and more stricter., for decis residual in agricultural product, there is stricter detection mark in China
Accurate
Method for deltamethrin pesticide detection is mainly gas chromatography, Normal-phase HPLC at present
Method and miniature chromatographic column purify capillary gas chromatography.But it is long all to there is the detection time in these methods, pre-treatment
Complexity, consumes a large amount of solvent, needs the shortcomings such as expensive instrument, and can not carry out on-the-spot on-line checking or follow the tracks of prison
Survey.Therefore, a kind of easy in the urgent need to invention, quickly, sensitive detection method.
Enzyme Linked Immunoadsorbent Assay, i.e. ELISA method.It utilizes the immunity of enzyme marker synantigen antibody complex instead
Should combine with the catalytic amplification of enzyme, i.e. maintain the sensitivity of enzymic catalytic reaction, maintain again antigen and resist
The specificity of body, thus greatly improve sensitivity.It is again a kind of heterogeneous immunoassay simultaneously, i.e. exists
Each step in reaction has washing process, thus eliminates and do not reflect material and interfering material.
Hapten refers to, can occur with corresponding antibodies antigen-antibody reaction, again can not independent excitation human or animal
Body produces the antigen of antibody.It only has reactionogenicity, does not have immunogenicity, also known as incomplete antigen.Great majority
Polysaccharide and all of lipoid broadly fall into hapten.
Generally, there is immunogenic molecular weight of material and should be greater than 10000, and the molecular weight of pesticide is the least
In 1000, body typically can not be stimulated to produce the specific antibody for pesticide antigenic determinant, it is therefore necessary to
With just there is after macromolecular carrier coupling immunogenicity, this be also small molecule immune analyze basic model.Half is anti-
The selectivity of former structure antagonist is most important, and the design of hapten molecule and synthesis are small molecule immune analyses
The key point of technology.The design of hapten molecule should be with pesticide parent and the metabolite having toxicological significance
Molecular structure be target compound, for determined to as if single pesticide or a certain class pesticide, in design should
Correspondingly highlight basic molecular framework (i.e. parent nucleus) total in the molecular structure of specific pesticide or a class pesticide,
Synthesize corresponding single specificity antibody and group-specific antibody.Usual to the pesticide molecule not possessing active group
With direct derivatization or with Material synthesis, it is also possible to pesticide metabolism to be measured or catabolite are as hapten.Right
In the pesticide that some molecule is the least and unstable, the hapten of its derivatization can be synthesized.General preferable hapten
Molecule should possess the stereochemical characteristics similar with determinand as far as possible, to reduce immune cross-reactivity, improves anti-
The specificity of body；Simultaneously, it is ensured that after hapten is compound with the macromole of carrier, haptenic feature structure energy
Identified, to prepare, there is expection selectivity and the antibody of high-affinity to greatest extent by immunologically competent cell.
Artificial antigen is the complete antigen that hapten obtains with macromolecular carrier albumen coupling.Using carrier is not simply
Increase haptenic molecular weight, it is often more important that utilize its stronger immunogenicity induction body to produce immunne response,
So that body produces for hapten and the specific antibody of artificial antigen.
Immuno analytical method it is crucial that haptenic MOLECULE DESIGN, synthesis and artificial holoantigen and the preparation of antibody.
Therefore, target analyte molecule immunological characteristic, and how to highlight by chemistry or biochemical technology and to utilize this
A little characteristics, are the important research contents in this field.This technology has become as the brand-new field of microanalysis research,
A new analysis approach can be become side by side with traditional analysis.This type of porcess for artificial antigen of cyanobromide chrysanthemum ester and specificity
Antibody and set up immune analysis method based on this there is not been reported.
Patent application 200810153758.3 is the preparation about pesticide deltamethrin artificial semiantigen and antibody,
Employ structural formula
Hapten, prepare artificial antigen, then prepare antibody, enzyme labelled antibody.Refer in the patent for pesticide
The immune detection of decis, but the method not illustrating use.The most do not use this artificial anti-
Former and antibody is used for the concrete grammar of the detection of pesticide deltamethrin.
Summary of the invention
For above-mentioned situation, it is badly in need of a kind of simple and fast at present and effectively detects the method that pesticide deltamethrin is molten.
It is an object of the invention to, by use, there is the hapten of pesticide deltamethrin of dibromo chrysanthemic acid structure and artificial anti-
Former, quickly detect the content of pesticide deltamethrin；Feature is that detection speed is fast, and detection is accurately.
The technical solution used in the present invention is:
A kind of test kit for detecting decis, described test kit is by reagent set and is coated plate and forms, institute
The reagent set stated includes:
(1) PBS is concentrated: 40mM phosphate buffer, pH7.4, steams with double during use
The dilution of 5 times of water；
(2) chromogenic substrate: tetramethyl benzidine-hydrogen peroxide solution；
(3) concentrated cleaning solution: containing the phosphate buffered solution of 1% tween 20, distilled water during use
5 times of dilutions；
(4) sulphuric acid of stop buffer: 1.25M；
(5) decis standard solution: its decis standard solution mother solution is 1mg/mL, during use
After being diluted to 100 μ g/mL with PBS buffer solution, then press 1:4 6 gradients of dilution；
(6) decis enzyme-labelled antigen: decis hapten and the conjugate of horseradish peroxidase,
100,000 times are diluted with PBS during use；
Described be coated plate be the antibody prepared is dissolved in 50mmol, pH9～10 carbonic acid buffer in, join
Making the liquid that is coated of 10mg/ml, the every hole of ELISA Plate adds 100 μ L and is coated liquid, and room temperature stands 12-16 hour,
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of ELISA Plate will be coated again) wash 3 times；
The every hole of ELISA Plate adds 200 μ L, the BSA/PBS confining liquid of 1%, close one hour, then will close
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of ELISA Plate) wash liquid 3 times, after lyophilizing
Vacuum packaging, in 4 DEG C of preservations.
The antibody of above-mentioned use uses following steps to prepare:
Immunity: immune animal uses Male New Zealand large ear rabbit, immunization method uses subcutaneous and intramuscular injection,
Carry out 6 immunity altogether, booster immunization after initial immunity the 2nd week respectively, within the 4th week and the 6th week, carry out three times, with
It is for latter twice to carry out once for one month, after the 3rd immunity, starts blood sampling survey titer, after blood sampling time is immunity
10th day；
Initial immunity: take 1mg artificial antigen be dissolved in 0.9% NaCL and Fu Shi Freund's complete adjuvant equal-volume preparation
In solution, carry out animal immune；
Booster immunization: take 0.5mg artificial antigen and be dissolved in NaCL and the Fu Shi Freund's incomplete adjuvant equal-volume preparation of 0.9%
Solution in, carry out animal immune；
Antibody purification: timing detection animal antibody titer, when antibody reaches suitable titer to certain envelope antigen
Time, gather blood, and the centrifugal antiserum that obtains, use G-Sepharose albumen affinity column antagonistic Serum
It is purified, prepares IgG antibody；
Artificial antigen described above adopts and prepares with the following method:
(1) synthesis 6-aminocaprolc acid methyl ester
Reaction equation:
Weigh 400～410mg aminocaproic acids and be dissolved among 4mL methanol, stir under ice bath, be slowly added dropwise protochloride
Sulfone, continues to stir under ice bath 30min～40min to insoluble matter after being completely dissolved, after sloughing solvent under decompression and get final product
AME；
(2) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-methyl caproate
Reaction equation:
Weighing dibromo chrysanthemic acid 80～82mg to be dissolved in equipped with in 6mL anhydrous methylene chloride, ice bath stirring is reacted
30min；Sequentially add 1.0～2.0mgDMAP and 69～72mgDCC, under nitrogen protection, be stirred at room temperature 4～5h；
Adding 43～44mgAME, stirring, reaction is overnight；Filter off in reactant liquor and precipitate, then through 0.1mol/L hydrochloric acid,
Saturated sodium bicarbonate, distilled water and saturated nacl aqueous solution washing, finally decompression is removed solution, is obtained DAE；
(3) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-caproic acid
Reaction equation:
Weigh 128～130mgDAE to be dissolved in equipped with in 1.62mL ethanol, drip 2.3mL while stirring,
2mol/LNaOH, is heated to reflux 3h, and then decompression removes ethanol, is acidified to pH=3 with hydrochloric acid, and liquid is solid
Changing, add chloroform and make it dissolve, then wash away contained a small amount of ethanol with distilled water, decompression is removed solvent and get final product
Pesticide deltamethrin hapten；
(4) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-dimethyl-cyclopropane carbonyl]-amino }-caproic acid-2,5-dioxypyrrole
Alkane-1-base ester
Reaction equation:
Weigh 72～73mgDA and 25～26mgNHS and be dissolved in the anhydrous THF of 2.2mL, add under nitrogen protection
Enter 45～46mgDCC；Room temperature reaction is overnight；Filter, slough solvent under decompression, obtain pesticide deltamethrin
Haptenic Acibenzolar；
(5) synthesis of artificial antigen
The haptenic Acibenzolar 1.6mg taking said method synthesis is dissolved in 2ml dimethylformamide wiring solution-forming A,
0.86mg hemocyanin is wiring solution-forming B in the 0.2mol/L phosphate buffer solution of 2ml PH7.4, then at ice
Under bath, solution A is added in solution B slowly, agitated after react overnight at 4 DEG C, finally by reactant liquor
Inserting in bag filter, dialysing with the 0.2mol/L phosphate buffer solution of pH=7.4 at 4 DEG C i.e. can get people in three days
Work antigen；
The preparation method of described enzyme-labelled antigen is: the hapten Acibenzolar 1.9mg taking said method synthesis is dissolved in 2ml
Dimethylformamide wiring solution-forming A, 0.54mg horseradish peroxidase is dissolved in the 0.2mol/L phosphorus of 2ml pH7.4
Wiring solution-forming B in acid buffer, then adds solution A in solution B under ice bath slowly, agitated after
React overnight at 4 DEG C, finally reactant liquor is inserted in bag filter, with the 0.2mol/L of pH7.4 at 4 DEG C
Phosphate buffer dialysis i.e. can get enzyme-labelled antigen, for chromogenic reaction in three days；
The haptenic structure of the present invention is:
Hapten is connected formation antigen with carrier protein
Hapten is connected formation enzyme-labelled antigen with HRP
Pesticide deltamethrin hapten synthesis technology path:
(1) synthesis 6-aminocaprolc acid methyl ester (AME)
Reaction equation:
Weigh (400～410mg) aminocaproic acid and be dissolved among 4mL methanol, stir under ice bath, slowly drip
Add thionyl chloride, continue to stir under ice bath 30min～40min after being completely dissolved to insoluble matter, slough molten under decompression
AME is i.e. obtained after agent.
(2) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-methyl caproate (DAE)
Reaction equation:
Weigh dibromo chrysanthemic acid (80～82mg) and be dissolved in equipped with in 6mL dichloromethane (anhydrous), ice bath
Stirring reaction 30min；Sequentially adding (1.0～2.0mg) DMAP and (69～72mg) DCC, nitrogen is protected
Protect down and be stirred at room temperature 4～5h；Adding (43～44mg) AME, stirring, reaction is overnight.It is heavy to filter off in reactant liquor
Form sediment, then through 0.1mol/L hydrochloric acid, saturated sodium bicarbonate, distilled water and saturated nacl aqueous solution washing, finally subtract
Pressure removes solution, obtains DAE.
(3) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-caproic acid (DA)
Reaction equation:
Weigh (128～130mg) DAE and be dissolved in equipped with in 1.62mL ethanol, dropping while stirring (2.3mL,
2mol/L) NaOH, is heated to reflux 3h.Then decompression removes ethanol, is acidified to pH=3 with hydrochloric acid, and liquid is solid
Changing, add chloroform and make it dissolve, then wash away contained a small amount of ethanol with distilled water, decompression is removed solvent and get final product
Pesticide deltamethrin hapten DA.
(4) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-dimethyl-cyclopropane carbonyl]-amino }-caproic acid-2,5-dioxy
Pyrrolidin-1-yl ester (DHAE)
Reaction equation:
It is anhydrous that weighing (72～73mg) DA and (25～26mg) NHS is dissolved in 2.2mL THF() dissolve,
Add (45～46mg) DCC under nitrogen protection；Room temperature reaction is overnight；Filter, under decompression, slough solvent,
Obtain the Acibenzolar of pesticide deltamethrin hapten DA.
The invention also discloses described test kit for detecting the content of decis, comprise the steps:
(1) pre-treatment of sample under room temperature；
(2) before detection, being opened by test kit, it is little that all reagent and specimen all must at room temperature balance 1-2
Time；
(3) sample-adding and competitive reaction: the determinand processing 30 minutes with NaOH is dissolved in PBS and ladder
Degree dilution, decis enzyme-labelled antigen also is soluble in PBS, and during sample-adding, to add 100 μ L to be measured in ELISA Plate every hole
Thing and decis enzyme standard solution, competitive reaction 1 hour；
(4) colour developing: chromogenic substrate uses tetramethyl benzidine-hydrogen peroxide solution, and the every hole of ELISA Plate adds 150
μ L tetramethyl benzidine-hydrogen peroxide solution, after developing the color 20 minutes, every hole adds the sulphuric acid of 50 μ L15mol/L eventually
Only, reactant liquor is reading in automatic microplate reader.
Further, described determinand extract, preparation method comprises the steps:
(1) sample weigh 2.50g in 50mL tool plug conical flask in, add 10mL n-hexane/acetone mixing
Extracting solution, mixed proportion is 97.5: 2.5, (V/V), is placed in the shaking table that temperature is 40～45 DEG C vibration 40
Min, the extracting solution of system；
(2) taking 5mm × 70mm glass chromatography column, tripoli in fluorine sieve of built-in 500mg, stigma adds 15mg
Activated carbon, by 5.0mL petrol ether/ethyl acetate mixed liquor, mixed proportion 9: 1(V/V) eluent
Pre-drip washing pillar；
(3) draw 1.0mL step (1) extracting solution and inject chromatographic column, by the petroleum ether/second of 25.0mL
Acetoacetic ester mixed liquor mixed proportion 9: 1(V/V) eluting, collect leacheate；
(4) it is concentrated into 2-3mL with high pure nitrogen purging, adds 2.0mL normal hexane and purge again and be concentrated into 1.0mL,
Make final sample extracting solution.
The invention has the beneficial effects as follows: artificial antigen that the present invention uses specific hapten to prepare and antibody for
Detection pesticide deltamethrin, detection speed is fast, and detection is accurately.
Accompanying drawing illustrates:
Fig. 1 is the standard curve of decis
Detailed description of the invention:
Embodiment 1
1, the synthesis of artificial antigen
(1) synthesis 6-aminocaprolc acid methyl ester (AME)
Reaction equation:
Weigh 400 aminocaproic acids and be dissolved among 4mL methanol, stir under ice bath, be slowly added dropwise thionyl chloride,
Continue after being completely dissolved to insoluble matter to stir 30min under ice bath, after sloughing solvent under decompression, i.e. obtain AME.
(2) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-methyl caproate (DAE)
Reaction equation:
Weighing dibromo chrysanthemic acid 80 to be dissolved in equipped with in 6mL dichloromethane (anhydrous), 30min is reacted in ice bath stirring；
Sequentially add 1.0mgDMAP and 70mgDCC, under nitrogen protection, be stirred at room temperature 4～5h；Add
43mgAME, stirring, reaction is overnight.Filter off in reactant liquor and precipitate, then through 0.1mol/L hydrochloric acid, saturated carbon
Acid hydrogen sodium, distilled water and saturated nacl aqueous solution washing, finally decompression is removed solution, is obtained DAE.
(3) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-caproic acid (DA)
Reaction equation:
Weigh 128mgDAE to be dissolved in equipped with in 1.62mL ethanol, drip (2.3mL, 2mol/L) while stirring
NaOH, is heated to reflux 3h.Then decompression removes ethanol, is acidified to pH=3 with hydrochloric acid, liquid curing, then
Adding chloroform makes it dissolve, and then washes away contained a small amount of ethanol with distilled water, and decompression is removed solvent and i.e. obtained pesticide bromine
Cyano chrysanthemate hapten DA.
(4) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-dimethyl-cyclopropane carbonyl]-amino }-caproic acid-2,5-dioxypyrrole
Alkane-1-base ester (DHAE)
Reaction equation:
It is anhydrous that weighing 72mgDA and 25mgNHS is dissolved in 2.2mL THF() dissolve, under nitrogen protection
Add 45mgDCC；Room temperature reaction is overnight；Filter, under decompression, slough solvent, obtain pesticide deltamethrin half
The Acibenzolar of antigen DA.
(5) synthesis of artificial antigen
The haptenic Acibenzolar 1.6mg taking said method synthesis is dissolved in 2ml dimethylformamide wiring solution-forming A,
0.86mg hemocyanin is wiring solution-forming B in the 0.2mol/L phosphate buffer solution of 2ml PH7.4, then
Under ice bath, solution A is added in solution B slowly, agitated after react overnight at 4 DEG C, finally will
Reactant liquor is inserted in bag filter, dialyses three days with the 0.2mol/L phosphate buffer solution of pH=7.4 at 4 DEG C
I.e. can get artificial antigen；
2, the preparation of antibody
Immunity: immune animal uses Male New Zealand large ear rabbit, immunization method uses subcutaneous and intramuscular injection, altogether
Carry out 6 immunity, booster immunization after initial immunity the 2nd week respectively, within the 4th week and the 6th week, carry out three times, after
Being for twice to carry out once for one month, start blood sampling and survey titer after the 3rd immunity, blood sampling time is after immunity the
10 days；
Initial immunity: take 1mg artificial antigen be dissolved in 0.9% NaCL and Fu Shi Freund's complete adjuvant equal-volume preparation
In solution, carry out animal immune；
Booster immunization: take 0.5mg artificial antigen and be dissolved in NaCL and the Fu Shi Freund's incomplete adjuvant equal-volume preparation of 0.9%
Solution in, carry out animal immune；
Antibody purification: timing detection animal antibody titer, when antibody reaches suitable titer to certain envelope antigen
Time, gather blood, and the centrifugal antiserum that obtains, use G-Sepharose albumen affinity column antagonistic Serum
It is purified, prepares IgG antibody；
The preparation of 3 enzyme-labelled antigens
The hapten Acibenzolar 1.9mg taking said method synthesis is dissolved in 2ml dimethylformamide wiring solution-forming A,
0.54mg horseradish peroxidase is dissolved in wiring solution-forming B in the 0.2mol/L phosphate buffer of 2ml pH7.4,
Then under ice bath, solution A is added in solution B slowly, agitated after react overnight at 4 DEG C,
Finally reactant liquor is inserted in bag filter, with the 0.2mol/L phosphate buffer dialysis three of pH7.4 at 4 DEG C
It i.e. can get enzyme-labelled antigen, for chromogenic reaction.
The preparation of embodiment 2 test kit
A kind of test kit for detecting decis, by reagent set be coated plate and form, described reagent set bag
Include:
(1) PBS is concentrated: 40mM phosphate buffer, pH7.4, steams with double during use
The dilution of 5 times of water；
(2) chromogenic substrate: tetramethyl benzidine-hydrogen peroxide solution；
(3) concentrated cleaning solution: containing the phosphate buffered solution of 1% tween 20, distilled water during use
5 times of dilutions；
(4) sulphuric acid of stop buffer: 1.25M；
(5) decis standard solution: its decis standard solution mother solution is 1mg/mL, during use
After being diluted to 100 μ g/mL with PBS buffer solution, then press 1:4 6 gradients of dilution；
(6) decis enzyme-labelled antigen: decis hapten and the conjugate of horseradish peroxidase,
100,000 times are diluted with PBS during use；
Described be coated plate be the antibody prepared is dissolved in 50mmol, pH9～10 carbonic acid buffer in, join
Making the liquid that is coated of 10mg/ml, the every hole of ELISA Plate adds 100 μ L and is coated liquid, and room temperature stands 12 hours, then
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of ELISA Plate will be coated) wash 3 times；Enzyme
The every hole of target adds 200 μ L, the BSA/PBS confining liquid of 1%, closes one hour, then the enzyme that will have closed
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of target) wash liquid 3 times, true after lyophilizing
Empty package, in 4 DEG C of preservations.
The preparation of embodiment 3 test kit
A kind of test kit for detecting decis, it is characterised in that: described test kit is by reagent set and bag
Being formed by plate, described reagent set includes:
(1) PBS is concentrated: 40mM phosphate buffer, pH7.4, steams with double during use
The dilution of 5 times of water；
(2) chromogenic substrate: tetramethyl benzidine-hydrogen peroxide solution；
(3) concentrated cleaning solution: containing the phosphate buffered solution of 1% tween 20, distilled water during use
5 times of dilutions；
(4) sulphuric acid of stop buffer: 1.25M；
(5) decis standard solution: its decis standard solution mother solution is 1mg/mL, during use
After being diluted to 100 μ g/mL with PBS buffer solution, then press 1:4 6 gradients of dilution；
(6) decis enzyme-labelled antigen: decis hapten and the conjugate of horseradish peroxidase,
100,000 times are diluted with PBS during use；
Described be coated plate be the antibody prepared is dissolved in 50mmol, pH9～10 carbonic acid buffer in, join
Making the liquid that is coated of 10mg/ml, the every hole of ELISA Plate adds 100 μ L and is coated liquid, and room temperature stands 16 hours, then
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of ELISA Plate will be coated) wash 3 times；Enzyme
The every hole of target adds 200 μ L, the BSA/PBS confining liquid of 1%, closes one hour, then the enzyme that will have closed
Phosphate buffered solution 0.05%(v/v of every hole polysorbas20 of target) wash liquid 3 times, true after lyophilizing
Empty package, in 4 DEG C of preservations.
Embodiment 4 test kit is for detecting the content of decis in Folium Camelliae sinensis
(1) pre-treatment of Folium Camelliae sinensis
(1) Tea Samples weigh 2.50g in 50mL tool plug conical flask in, add 10mL n-hexane/acetone
Mixed extract, mixed proportion is 97.5: 2.5, (V/V), is placed in the shaking table that temperature is 40～45 DEG C and shakes
Swing 40min, the extracting solution of system；
(2) taking 5mm × 70mm glass chromatography column, tripoli in fluorine sieve of built-in 500mg, stigma adds 15mg
Activated carbon, by 5.0mL petrol ether/ethyl acetate mixed liquor, mixed proportion 9: 1(V/V) eluent
Pre-drip washing pillar；
(3) draw 1.0mL step (1) extracting solution and inject chromatographic column, by the petroleum ether/second of 25.0mL
Acetoacetic ester mixed liquor mixed proportion 9: 1(V/V) eluting, collect leacheate；
(4) it is concentrated into 2-3mL with high pure nitrogen purging, adds 2.0mL normal hexane and purge again and be concentrated into 1.0mL,
Make final sample extracting solution.
(2) before detection, being opened by test kit, all reagent and specimen all must at room temperature balance 1-2 hour；
(3) sample-adding and competitive reaction: the determinand processing 30 minutes with NaOH is dissolved in PBS and gradient is dilute
Releasing, decis enzyme-labelled antigen also is soluble in PBS, during sample-adding the every hole of ELISA Plate add 100 μ L determinands and
Decis enzyme standard solution, competitive reaction 1 hour；
(4) colour developing: chromogenic substrate uses tetramethyl benzidine-hydrogen peroxide solution, and the every hole of ELISA Plate adds 150 μ L tetra-
Methyl biphenyl amine-hydrogen peroxide solution, after developing the color 20 minutes, every hole adds the sulphuric acid termination of 50 μ L15mol/L, instead
Answer liquid reading in automatic microplate reader.
Embodiment 5 test kit is for detecting the content of decis in vegetable
(1) pre-treatment of vegetable
(1) vegetable sample weigh 2.50g in 50mL tool plug conical flask in, add 10mL n-hexane/acetone
Mixed extract, mixed proportion is 97.5: 2.5, (V/V), is placed in the shaking table that temperature is 40～45 DEG C and shakes
Swing 40min, the extracting solution of system；
(2) taking 5mm × 70mm glass chromatography column, tripoli in fluorine sieve of built-in 500mg, stigma adds 15mg
Activated carbon, by 5.0mL petrol ether/ethyl acetate mixed liquor, mixed proportion 9: 1(V/V) eluent
Pre-drip washing pillar；
(3) draw 1.0mL step (1) extracting solution and inject chromatographic column, by the petroleum ether/second of 25.0mL
Acetoacetic ester mixed liquor mixed proportion 9: 1(V/V) eluting, collect leacheate；
(4) it is concentrated into 2-3mL with high pure nitrogen purging, adds 2.0mL normal hexane and purge again and be concentrated into 1.0mL,
Make final sample extracting solution.
(2) before detection, being opened by test kit, all reagent and specimen all must at room temperature balance 1-2 hour；
(3) sample-adding and competitive reaction: the determinand processing 30 minutes with NaOH is dissolved in PBS and gradient is dilute
Releasing, decis enzyme-labelled antigen also is soluble in PBS, during sample-adding the every hole of ELISA Plate add 100 μ L determinands and
Decis enzyme standard solution, competitive reaction 1 hour；
(4) colour developing: chromogenic substrate uses tetramethyl benzidine-hydrogen peroxide solution, and the every hole of ELISA Plate adds 150 μ L tetra-
Methyl biphenyl amine-hydrogen peroxide solution, after developing the color 20 minutes, every hole adds the sulphuric acid termination of 50 μ L15mol/L, instead
Answer liquid reading in automatic microplate reader.
Claims (1)
1., for detecting the test kit of decis, described test kit is by reagent set and is coated plate and forms, described
Reagent set includes:
(1) PBS is concentrated: 40mM phosphate buffer, pH 7.4, with distilled water 5 times dilution during use；
(2) chromogenic substrate: tetramethyl benzidine-hydrogen peroxide solution；
(3) concentrated cleaning solution: containing the phosphate buffered solution of 1% tween 20, with distilled water 5 times dilution during use；
(4) sulphuric acid of stop buffer: 1.25M；
(5) decis standard solution: its decis standard solution mother solution is 1mg/mL, buffers with PBS during use
After solution is diluted to 100 μ g/mL, then press 1:4 6 gradients of dilution；
(6) decis enzyme-labelled antigen: decis hapten and the conjugate of horseradish peroxidase, PBS during use
Buffer dilutes 100,000 times；
Described be coated plate be the antibody prepared is dissolved in 50mmol, pH9～10 carbonic acid buffer in, be configured to
10mg/ml is coated liquid, and the every hole of ELISA Plate adds 100 μ L and is coated liquid, and room temperature stands 12-16 hour, then will be coated enzyme
The phosphate buffered solution volume fraction of every hole polysorbas20 of target is 0.05%, washs 3 times；The every hole of ELISA Plate adds
200 μ L, the BSA/PBS confining liquid of 1%, close one hour, then by every hole polysorbas20 of ELISA Plate of having closed
Phosphate buffered solution volume fraction is 0.05% wash liquid 3 times, and lyophilizing final vacuum is packed, in 4 DEG C of preservations；
The antibody of above-mentioned use uses following steps to prepare:
Immunity: immune animal uses Male New Zealand large ear rabbit, immunization method uses subcutaneous and intramuscular injection, enters altogether
6 immunity of row, booster immunization after initial immunity the 2nd week respectively, within the 4th week and the 6th week, carry out three times, later twice is one
Within individual month, carrying out once, start blood sampling and survey titer after the 3rd immunity, blood sampling time is after immunity the 10th day；
Initial immunity: take 1mg artificial antigen and be dissolved in the solution of NaCL and Fu Shi Freund's complete adjuvant equal-volume preparation of 0.9%,
Carry out animal immune；
Booster immunization: take the solution that 0.5mg artificial antigen is dissolved in NaCL and the Fu Shi Freund's incomplete adjuvant equal-volume preparation of 0.9%
In, carry out animal immune；
Antibody purification: timing detection animal antibody titer, when antibody reaches suitable titer to envelope antigen, gathers blood,
And it is centrifuged acquisition antiserum, use G-Sepharose albumen affinity column antagonistic Serum to be purified, prepare IgG antibody；
Artificial antigen described above adopts and prepares with the following method:
(1) synthesis 6-aminocaprolc acid methyl ester (AME)
Reaction equation:
Weigh 400 aminocaproic acids and be dissolved among 4mL methanol, stir under ice bath, be slowly added dropwise thionyl chloride, to insoluble matter
Continue after being completely dissolved to stir 30min under ice bath, after sloughing solvent under decompression, i.e. obtain AME；
(2) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-methyl caproate (DAE)
Reaction equation:
Weighing dibromo chrysanthemic acid 80 to be dissolved in equipped with in 6mL anhydrous methylene chloride, 30min is reacted in ice bath stirring；Add the most successively
Enter 1.0mgDMAP and 70mgDCC, under nitrogen protection, be stirred at room temperature 4～5h；Add 43mgAME, stirring, reaction
Overnight, filter off in reactant liquor and precipitate, more molten through 0.1mol/L hydrochloric acid, saturated sodium bicarbonate, distilled water and saturated sodium-chloride
Liquid washs, and solution is removed in finally decompression, obtains DAE；
(3) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-Dimethyl-cyclopropyl carbonyl]-amino }-caproic acid (DA)
Reaction equation:
Weigh 128mgDAE to be dissolved in equipped with in 1.62mL ethanol, drip 2.3mL, 2mol/LNaOH while stirring, add
Hot reflux 3h；Then decompression removes ethanol, is acidified to pH=3, liquid curing with hydrochloric acid, adds chloroform and make it dissolve,
Then washing away contained a small amount of ethanol with distilled water, decompression is removed solvent and is i.e. obtained pesticide deltamethrin hapten DA；
(4) synthesis
6-{ [3-(2,2-dibromo vinyl)-2,2-dimethyl-cyclopropane carbonyl]-amino }-caproic acid-2,5-dioxypyrrole alkane-1-base ester
(DHAE)
Reaction equation:
Weigh 72mgDA and 25mgNHS and be dissolved in 2.2mL anhydrous THF dissolving, add 45mgDCC under nitrogen protection；
Room temperature reaction is overnight；Filter, slough solvent under decompression, obtain the Acibenzolar of pesticide deltamethrin hapten DA；
(5) synthesis of artificial antigen
The haptenic Acibenzolar 1.6mg taking said method synthesis is dissolved in 2ml dimethylformamide wiring solution-forming A,
0.86mg hemocyanin is wiring solution-forming B in the 0.2mol/L phosphate buffer solution of 2ml pH7.4, then under ice bath
Solution A is added in solution B slowly, agitated after react overnight at 4 DEG C, finally reactant liquor is inserted dialysis
In Dai, dialyse with the 0.2mol/L phosphate buffer solution of pH=7.4 at 4 DEG C and i.e. can get artificial antigen in three days；
The preparation method of described enzyme-labelled antigen is: the hapten Acibenzolar 1.9mg taking said method synthesis is dissolved in 2ml dimethyl
Methanamide wiring solution-forming A, 0.54mg horseradish peroxidase is dissolved in the 0.2mol/L phosphate buffer of 2ml pH 7.4 and being made into
Solution B, then adds solution A in solution B under ice bath slowly, agitated after react overnight at 4 DEG C, finally
Being inserted by reactant liquor in bag filter, dialysing with the 0.2mol/L phosphate buffer of pH7.4 at 4 DEG C i.e. can get enzyme mark in three days
Antigen, for chromogenic reaction, for monitoring the content of decis in vegetable, comprises the steps:
(1) pre-treatment of vegetable
(1) vegetable sample weigh 2.50g in 50mL tool plug conical flask in, add 10mL n-hexane/acetone mixed extract,
Mixed volume ratio is 97.5: 2.5, is placed in the shaking table that temperature is 40～45 DEG C vibration 40min, the extracting solution of system；
(2) taking 5mm × 70mm glass chromatography column, tripoli in fluorine sieve of built-in 500mg, stigma adds the activity of 15mg
Charcoal, with 5.0mL petrol ether/ethyl acetate mixed liquor, mixed volume ratio 9: 1 eluent pre-drip washing pillar；
(3) draw 1.0mL step (1) extracting solution and inject chromatographic column, mix by the petrol ether/ethyl acetate of 25.0mL
Close liquid mixture and amass ratio 9: 1 eluting, collect leacheate；
(4) it is concentrated into 2-3mL with high pure nitrogen purging, adds 2.0mL normal hexane and purge again and be concentrated into 1.0mL, make
Whole sample extracting solution；
(2) before detection, being opened by test kit, all reagent and specimen all must at room temperature balance 1-2 hour；
(3) sample-adding and competitive reaction: the determinand processing 30 minutes with NaOH is dissolved in PBS and gradient dilution, bromine
Cyano chrysanthemate enzyme-labelled antigen also is soluble in PBS, and during sample-adding, the every hole of ELISA Plate adds 100 μ L determinands and decis enzyme standard
Solution, competitive reaction 1 hour；
(4) colour developing: chromogenic substrate uses tetramethyl benzidine-hydrogen peroxide solution, and the every hole of ELISA Plate adds 150 μ L tetramethyls
Benzidine-hydrogen peroxide solution, after developing the color 20 minutes, every hole adds the sulphuric acid termination of 50 μ L 15mol/L, and reactant liquor is at automatic enzyme mark
Reading on instrument.
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| CN1948964A (en) * | 2006-11-27 | 2007-04-18 | 赵建庄 | Synthesizing porcess for artificial antigen of cyanobromide chrysanthemum ester and assaying process thereof |
| CN101339189A (en) * | 2008-08-18 | 2009-01-07 | 北京农学院 | Deltamethrin polyclonal antibody enzyme-linked immunity detection method and its uses |
| CN101747428A (en) * | 2008-12-05 | 2010-06-23 | 天津科技大学 | Specific antibody against pesticide deltamethrin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1948964A (en) * | 2006-11-27 | 2007-04-18 | 赵建庄 | Synthesizing porcess for artificial antigen of cyanobromide chrysanthemum ester and assaying process thereof |
| CN101339189A (en) * | 2008-08-18 | 2009-01-07 | 北京农学院 | Deltamethrin polyclonal antibody enzyme-linked immunity detection method and its uses |
| CN101747428A (en) * | 2008-12-05 | 2010-06-23 | 天津科技大学 | Specific antibody against pesticide deltamethrin |
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