CN108387728A - A kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion and detection method - Google Patents
A kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion and detection method Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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Abstract
The present invention relates to field of chemical detection, in particular to a kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion and detection method；The kit includes：Molecular imprinted polymer membrane ELISA Plate and Hostathion enzyme-labelled antigen；It is described to be prepared by the following method to obtain：Using triazolone as template molecule, prepolymerization is carried out in atent solvent with function monomer, crosslinking agent, initiator and obtain prepolymerization liquid；Will the prepolymerization liquid be added ELISA Plate in, vacuum drying polymerization after eluted template molecule to obtain the final product.Hostathion adsorption functional material provided by the invention have higher stability, the ability of longer service life and stronger anti-adverse environment, overcome traditional biological Antibody preparation period length, easy in inactivation, it is of high cost the shortcomings of.
Description
Technical field
The present invention relates to field of chemical detection, are tried in particular to a kind of bionical MBP enzyme linked immuno-adsorbent assay of Hostathion
Agent box and detection method.
Background technology
Hostathion (triazophos) is a kind of medium poison, broad-spectrum organophosphorous pesticide, has strong tag and stomach toxicity
Effect after entering human body with food, can cause a large amount of acetylcholines to be accumulated in neural effector junction, occur muscarinic,
Nicotine sample and central nervous system symptom were forbidden using Hostathion on vegetables from 31 days December in 2016.
The common detection methods of Hostathion have gas chromatography, ultra performance liquid chromatography-tandem mass spectrometry etc. at present, but
These method complex pretreatments, of high cost, detection speed is slow, is unsuitable for live detection in real time.Immunoassay has high special
Property, high sensitivity and incomparable highly selective of conventional physical and chemical analysis, play act in clinical detection and laboratory research
The effect of sufficient weight.But traditional biological antibody also shows that many deficiencies, such as micromolecular compound must synthesize half and resist
Original can just have immunogenicity, and preparation process is cumbersome, the period is long, need to sacrifice animal, and biological antibody physicochemical property is unstable,
Storage and use condition are stringent etc..Therefore design synthesis can compare favourably with biological antibody specificity is high, stability is good, it is low at
This bionic antibody has important practical significance.With the development of polymer chemistry and the appearance of molecular imprinting technology, molecule
Imprinted polymer becomes the ideal material of bionic antibody.MIP is the artificial synthesized high molecular material containing specific recognition site,
There are many advantages compared with biological antibody.Therefore molecular imprinting technology and ELISA are combined, prepare Hostathion point
Sub- blotting membrane establishes bionical enzyme-linked immunosorbent analytical technique and quickly detects Hostathion pesticide residue method, to study pollutant
Trace detection reference is provided, provide technical support for Hostathion contamination monitoring in agricultural product, and examine to current quickly analysis
One important supplement of survey technology research will push the development of Fast Determination of Pesticide Residue technology.
In view of this, special propose the present invention.
Invention content
That there are detection times it is an object of the invention to overcoming traditional Hostathion detection method is long, instrument price is expensive,
The defects of prepared by specific antibody difficulty, provides a kind of quick detection kit and method of Hostathion.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
The present invention relates to a kind of bionical enzyme-linked immuno sorbent assay kits of Hostathion, including：
Molecular imprinted polymer membrane ELISA Plate and Hostathion enzyme-labelled antigen；
It is described to be prepared by the following method to obtain：Using triazolone as template molecule, with function monomer, crosslinking agent, initiator
Prepolymerization is carried out in atent solvent obtains prepolymerization liquid；The prepolymerization liquid is added in ELISA Plate, after vacuum drying polymerization
Eluted template molecule to obtain the final product.
According to an aspect of the present invention, it is bionical using kit as described above progress Hostathion that the invention further relates to a kind of
The method of MBP enzyme linked immuno-adsorbent assay, including：
Blank control wells, negative control hole and detection hole are set on the molecular imprinted polymer membrane ELISA Plate；
Hostathion enzyme-labelled antigen dilution and Hostathion titer or sample extracting solution are added in the detection hole, and
The Hostathion titer or sample extracting solution being added in different holes are by gradient dilution；
Hostathion enzyme-labelled antigen dilution and buffer solution are added in the negative control hole；
Wherein, the Hostathion enzyme-labelled antigen dilution is diluted by Hostathion enzyme-labelled antigen standard solution with buffer solution
It arrives；
The buffer solution is added in the blank control wells；
Each hole is incubated at room temperature 50min~70min after being added in a manner described, bottom is separately added into each hole after board-washing
Thing liquid terminates reaction after being incubated at room temperature 30min~60min；Microplate reader reads each hole absorbance and calculates suppression according to the following formula
Rate processed：
IC%=[1- (ASample-ABlank)÷(AControl-ABlank)]×100；
In formula：
Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；
AControl--- the negative control hole mean absorbance values；
ASample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；
ABlank--- the mean absorbance values of the blank control wells；
Using the logarithm of Hostathion titer gradient dilution liquid or sample extracting solution gradient dilution liquid concentration as abscissa, phase
The inhibiting rate percentage answered is ordinate, draws standard curve respectively；
It is dense to get the sample extracting solution that the corresponding absorbance value of the sample extracting solution is substituted into the standard curve
Degree.
Compared with prior art, beneficial effects of the present invention are：
(1) Hostathion adsorption functional material provided by the invention has higher selectivity, can be solved using virtual template
Template leakage problems can replace biological antibody to be applied to Enzyme-multiplied immune technique；The adsorption functional material is prepared by chemical method,
Ability with higher stability, longer service life and stronger anti-adverse environment, overcomes traditional biological antibody system
Standby period length, easy in inactivation, it is of high cost the shortcomings of.
(2) of the invention to be combined molecular imprinting technology and Enzyme-multiplied immune technique, establish has high sensitivity to Hostathion
Bionic enzyme linked immunosorbent detection.The minimum detectability of this method is 7 μ gL-1, it is significantly less than maximum residue limit value,
It disclosure satisfy that detection needs.And analysis time (shortening 1.0h than traditional biological immunoassay) is substantially reduced, is suitable for fast
Speed detection Hostathion.
(3) bionic antibody prepared by the present invention may be reused 50 times or more, and cost substantially reduces；And pre-treatment letter
Single, high sensitivity, experimental implementation is simple, the quick detection of Hostathion suitable for various food.
Specific implementation mode
The present invention relates to a kind of bionical enzyme-linked immuno sorbent assay kits of Hostathion, including：
Molecular imprinted polymer membrane ELISA Plate and Hostathion enzyme-labelled antigen；
It is described to be prepared by the following method to obtain：Using triazolone as template molecule, with function monomer, crosslinking agent, initiator
Prepolymerization is carried out in atent solvent obtains prepolymerization liquid；The prepolymerization liquid is added in ELISA Plate, after vacuum drying polymerization
Eluted template molecule to obtain the final product.
Preferably, kit as described above, the function monomer are methacrylic acid, and the crosslinking agent is trimethoxy
Oxypropyl trimethyl acrylate, and the template molecule：Function monomer：The molar ratio of crosslinking agent is 1:5~7:2~4；
It is furthermore preferred that molar ratio is 1:6:3.
Preferably, kit as described above, the atent solvent are acetonitrile；It is furthermore preferred that the template molecule：Inertia
Solvent=1mol：20~30ml；More preferably 1mol：25ml.
Preferably, kit as described above, the initiator are azodiisobutyronitrile；It is furthermore preferred that the template point
Son：Initiator=1mol：40~60mg；More preferably 1mol：50mg.
Preferably, kit as described above, it is described vacuum drying polymerization condition be vacuum drying under the conditions of 30 DEG C~
50 DEG C of polymerization 12h~for 24 hours；
More preferably 40 DEG C polymerization 18h.
Preferably, the preparation method of kit as described above, the Hostathion enzyme-labelled antigen includes：
Hostathion haptens is synthesized into envelope antigen using mixed anhydride method, then presses the envelope antigen with zymoprotein
According to 18~22:1 molar ratio, more preferably 20:1 molar ratio is coupled；
Preferably, the zymoprotein is horseradish peroxidase.
Preferably, kit as described above, it is described that Hostathion haptens is synthesized into envelope antigen using mixed anhydride method
The step of specifically include：By Hostathion haptens and n,N-Dimethylformamide with 0.2mmol~0.3mmol：The ratio of 1ml
It is mixed to get mixed liquor, then positive tri-n-butylamine and ethyl chloroformate reaction 50min~70min are added into the mixed liquor, then will
Gained reaction solution is mixed with the CBS buffer solutions dissolved with OVA, after dialysis to obtain the final product；
It is furthermore preferred that described the step of Hostathion haptens is synthesized envelope antigen using mixed anhydride method, specifically includes：
By Hostathion haptens and n,N-Dimethylformamide with 0.25mmol：The ratio of 1ml is mixed to get mixed liquor, then to described
Positive tri-n-butylamine and ethyl chloroformate reaction 60min are added in mixed liquor, then gained reaction solution and the CBS bufferings dissolved with OVA is molten
Liquid mixes, after dialysis to obtain the final product.
Preferably, kit as described above, the kit further include buffer solution；It is furthermore preferred that the buffer solution is
PBS solution.
Preferably, kit as described above, the kit further include substrate solution；
The substrate solution is configured by substrate A and substrate B；
The ingredient of the substrate A includes：The anhydrous sodium acetate of 8.0g/L~8.4g/L, the β of 2.0g/L~3.0g/L-ring paste
The carbamide peroxide of essence and 140mg/L~160mg/L, pH=4.8~5.2；
The ingredient of the substrate B includes that ratio is 40mg~60mg:The 3,3,5,5- tetramethyl benzidines of 4ml~6ml:Two
Methyl sulfoxide；
It is furthermore preferred that the ingredient of the substrate A includes：The anhydrous sodium acetate of 8.2g/L, the beta-cyclodextrin of 2.5g/L and
The carbamide peroxide of 150mg/L, pH=5.0；
The ingredient of the substrate B includes that ratio is 50mg:The 3,3,5,5- tetramethyl benzidines of 5ml:Dimethyl sulfoxide (DMSO).
According to an aspect of the present invention, it is bionical using kit as described above progress Hostathion that the invention further relates to a kind of
The method of MBP enzyme linked immuno-adsorbent assay, including：
Blank control wells, negative control hole and detection hole are set on the molecular imprinted polymer membrane ELISA Plate；
Hostathion enzyme-labelled antigen dilution and Hostathion titer or sample extracting solution are added in the detection hole, and
The Hostathion titer or sample extracting solution being added in different holes are by gradient dilution；
Hostathion enzyme-labelled antigen dilution and buffer solution are added in the negative control hole；
Wherein, the Hostathion enzyme-labelled antigen dilution is diluted by Hostathion enzyme-labelled antigen standard solution with buffer solution
It arrives；
The buffer solution is added in the blank control wells；
Each hole is incubated at room temperature 50min~70min after being added in a manner described, bottom is separately added into each hole after board-washing
Thing liquid terminates reaction after being incubated at room temperature 30min~60min；Microplate reader reads each hole absorbance and calculates suppression according to the following formula
Rate processed：
IC%=[1- (ASample-ABlank)÷(AControl-ABlank)]×100；
In formula：
Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；
AControl--- the negative control hole mean absorbance values；
ASample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；
ABlank--- the mean absorbance values of the blank control wells；
Using the logarithm of Hostathion titer gradient dilution liquid or sample extracting solution gradient dilution liquid concentration as abscissa, phase
The inhibiting rate percentage answered is ordinate, draws standard curve respectively；
It is dense to get the sample extracting solution that the corresponding absorbance value of the sample extracting solution is substituted into the standard curve
Degree.
Preferably, the preparation method of method as described above, the sample extracting solution includes：
Buffer solution ultrasonic extraction is added after sample to be tested is crushed, filters to obtain the final product.
Preferably, the ratio of method as described above, the sample to be tested and the buffer solution is 1g~2g：5ml~
10ml。
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
A kind of bionical immunoassay method of Hostathion enzyme label is present embodiments provided, is included the following steps：
1. the synthesis of haptens：
(1) synthesis of O- ethyldichlorothiophosphates (TZM-1)
Weigh phosphorus thiochloride (PSCl3) 68g (about 0.4mol) is placed in the three-necked flask with low-reading thermometer, with cryosel
Water-bath is cooled to -10~-5 DEG C, is added dropwise to absolute ethyl alcohol 55g (about 1.2mol) with vigorous stirring, and stringent control is added dropwise
Speed makes reacting liquid temperature be not more than 0 DEG C always.After being added dropwise at 10 DEG C the reaction was continued 2h.Reaction is finished, with (0 ± 5) DEG C
Distilled water washing reaction liquid (100ml × 2), separates oil reservoir and with anhydrous Na2SO4It is dry, then be evaporated under reduced pressure through water pump, collect 65
DEG C~75 DEG C of fractions, obtain 51.8g colourless transparent oil liquids (yield 72.3%, in terms of phosphorus thiochloride).
(2) the O- ethyls-O- [synthesis of one chlorine (TZM-2) of 3- (1- phenyl -1,2,4- triazolyls) thiophosphoryl
It weighs TZM-1 36g (about 0.2mol) to be placed in 250ml three-necked flasks, is added with stirring about 16g (about 0.1mol).
1- phenyl -1,2,4- Triadimenols, adds 15mlTEA and 80mlDCM.After solid all dissolving, with ice-water bath by the solution
20 DEG C are cooled to hereinafter, trace catalyst is added and the NaOH aqueous solutions of 55ml 2mol/L are added dropwise, the reaction was continued 1h.Instead
After answering, 50ml5%NaOH ice water solutions are added, water layer are separated after oscillation, oil reservoir is washed with ice water to neutrality, then through anhydrous
Na2SO4 is dried, and is concentrated under reduced pressure, is obtained a small amount of brown oil, and with petroleum ether extraction (50ml × 2), extracting solution subtracts the grease again
Pressure concentration, obtains 10.6g yellow liquids (yield 35%, in terms of Triadimenol).
(3) synthesis of Hostathion haptens
1.03g (about 10mmol) 4-Aminobutanoicacid is weighed, is dissolved in 10mlNaOH solution (1mol/L), ice-water bath cooling
It is slowly added to the TZM-2 that 1.51g (about 5mmol) is dissolved in 10ml dioxane to 0~10 DEG C, under stirring, micro catalysis is added
Agent, then it is added dropwise to 10mlNaOH aqueous solutions (1mol/L).15~25 DEG C are warming up to, 4h is reacted.After completion of the reaction, 50ml is added
Water, then with petroleum ether reaction mixture (40ml × 2), petroleum ether layer is abandoned, it is about 3 to adjust water phase pH with 2mol/L HCL,
(40ml × 2) are extracted with ethyl acetate again, extracting solution is dried after being washed with a small amount through anhydrous Na 2SO4, is concentrated under reduced pressure, remaining
Overnight in 4 DEG C of sealing storages, whether there is or not the precipitations of chromaticity body for object.With acetic acid second it is cruel-petroleum ether recrystallization, filter, it is dry, obtain 0.52g
White solid (THBu, yield 27%, in terms of intermediate TZM-2).
2. the preparation of enzyme-labelled antigen
Haptens 0.25mmol is taken, 1mlDMF is dissolved in, is added with stirring the positive tri-n-butylamines of 60 μ l and 30 μ l ethyl chloroformates, room
Temperature is stirred to react 1h.Extract reaction solution the CBS buffer solutions (0.01mol/L) that 300 μ l are slowly dropped to 6ml dissolved with 120mgOVA
In, after reaction is stirred at room temperature, it is packed into bag filter, is first dialysed 3 times with distilled water, then uses the PBS dialysis 3d of 0.01mol/L, often
It changes dialyzate 3-4 times.Packing is taken out to be stored in -20 DEG C of refrigerator.
Antigen is coupled to enzyme egg from upper, a concentration of 10mg/ml (antigen and the zymoprotein of zymoprotein in reaction solution after being activated
Molar ratio be 20:1, enzyme is horseradish peroxidase).Isometric glycerine, mixing, packing, -20 DEG C of guarantors are added after having reacted
It deposits.
5 times of PBS solution groups are divided into：By Na2HPO4·12H2O：68.80g、NaH2PO4·2H2O：8.97g and NaCl：
45.00g adds distilled water to be settled to 1L compositions；It is PBS solution that 5 times of PBS distilled waters, which are diluted 5 times,.
3. the preparation of molecular imprinted polymer membrane
Triazolone (being dissolved in 20ml acetonitriles), methacrylic acid and trimethoxy oxypropyl trimethyl acrylate are according to 1:6:3
Molar ratio mixes, and 40mg azodiisobutyronitriles are then added, are stirred to react 0.5h.It is added on 200 μ l per hole in 96 hole elisa Plates
Mixed liquor is stated, 50 DEG C of polymerisation 12h under the conditions of vacuum drying, with 8:1 (v/v) methanol/glacial acetic acid solution elutes 8h, and methanol is molten
Liquid elutes 3h to remove triazolone, and molecular imprinted polymer membrane is obtained after dry.
4. residual enzyme more than marks the foundation of bionical immunoassay method
Imprinted polymer film prepared by step 3 is as bionic antibody；Enzyme-labelled antigen standard solution PBS prepared by step 2
Solution dilutes 5000 times, as enzyme mark dilution.
The 1st row of 96 hole elisa Plates is set as blank group, only adds 200 μ L PBS solutions per hole；2nd row is set as control group, often
Hole adds 100 μ L enzyme mark dilutions and 100 μ LPBS solution；3-8 rows are separately added into Hostathion titer successively per hole or sample carries
Take liquid gradient dilution liquid and 100 μ L enzyme mark dilutions.Phosphate Tween buffer PBST board-washings are used after being incubated at room temperature 1h five times.96
100-200 μ L substrate solutions are added per hole for hole elisa Plates, react 30-60min at room temperature.50-100 μ L1.25mol/L are added per hole
Sulfuric acid terminates reaction；96 hole elisa Plates 1-8 row absorbance value A are read with microplate reader, calculate separately inhibiting rate；
Inhibiting rate value of the various concentration Hostathion to antigen-antibody binding reaction is calculated according to the following formula：
IC%=[1- (ASample-ABlank)÷(AControl-ABlank)]×100；
In formula：
Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；
AControl--- the negative control hole mean absorbance values；
ASample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；
ABlank--- the mean absorbance values of the blank control wells；
Using the logarithm of Hostathion standard specimen gradient dilution liquid concentration as abscissa, corresponding inhibiting rate percentage is vertical sit
Mark, draws standard curve respectively.
The group of 5 times of PBST solution is divided into：200mL, 10% Tween-20：5mL adds distilled water to be settled to 1L；By 5
It is PBST solution that times PBST distilled water, which dilutes 5 times,.
The substrate solution is formulated as：Substrate A：8.2g anhydrous sodium acetates, 2.5g beta-cyclodextrins and 150mg hydrogen peroxide
Urea adds distilled water to be settled to 1000mL, adjusts pH to 5.0,4 DEG C of preservations；Substrate B：50mg 3,3,5,5- tetramethyl benzidines are dissolved in
5mL dimethyl sulfoxide (DMSO)s, room temperature are kept in dark place；Using preceding 15min, 14.6mL substrate As and 0.45mL substrate Bs is taken to be mixed into substrate
Liquid.
5. weighing 1g samples to be tested, 5mL PBS solutions ultrasonic extraction is added after crushing 3 times, filters to obtain sample extracting solution.It will
Sample extracting solution replaces Hostathion standard specimen gradient dilution liquid described in step 4), step 4) operation is repeated, according to inhibiting rate by marking
Directrix curve calculates the content of Hostathion in determinand.
Embodiment 2
A kind of bionical immunoassay method of Hostathion enzyme label is present embodiments provided, is included the following steps：
Step 1 is the same as embodiment 1.
2. the preparation of enzyme-labelled antigen
Haptens 0.3mmol is taken, 1mlDMF is dissolved in, is added with stirring the positive tri-n-butylamines of 60 μ l and 30 μ l ethyl chloroformates, room
Temperature is stirred to react 1h.Extract reaction solution the CBS buffer solutions (0.01mol/L) that 300 μ l are slowly dropped to 6ml dissolved with 120mgOVA
In, after reaction is stirred at room temperature, it is packed into bag filter, is first dialysed 3 times with distilled water, then uses the PBS dialysis 3d of 0.01mol/L, often
It changes dialyzate 3-4 times.Packing is taken out to be stored in -20 DEG C of refrigerator.
Antigen is coupled to enzyme egg from upper, a concentration of 10mg/ml (antigen and the zymoprotein of zymoprotein in reaction solution after being activated
Molar ratio be 20:1, enzyme is horseradish peroxidase).Isometric glycerine, mixing, packing, -20 DEG C of guarantors are added after having reacted
It deposits.
5 times of PBS solution groups are divided into：By Na2HPO4·12H2O：68.80g、NaH2PO4·2H2O：8.97g and NaCl：
45.00g adds distilled water to be settled to 1L compositions；It is PBS solution that 5 times of PBS distilled waters, which are diluted 5 times,.
3. the preparation of molecular imprinted polymer membrane
Triazolone (being dissolved in 30ml acetonitriles), methacrylic acid and trimethoxy oxypropyl trimethyl acrylate are according to 1:5:4
Molar ratio mixes, and 60mg azodiisobutyronitriles are then added, are stirred to react 1.5h.It is added on 200 μ l per hole in 96 hole elisa Plates
State mixed liquor, under the conditions of vacuum drying 30 DEG C of polymerisations for 24 hours, with 7:1 (v/v) methanol/glacial acetic acid solution elutes 15h, methanol
Solution elutes 5h to remove triazolone, and molecular imprinted polymer membrane is obtained after dry.
4. residual enzyme more than marks the foundation of bionical immunoassay method
Imprinted polymer film prepared by step 3 is as bionic antibody；Enzyme-labelled antigen standard solution PBS prepared by step 2
Solution dilutes 5000 times, as enzyme mark dilution.
The 1st row of 96 hole elisa Plates is set as blank group, only adds 200 μ L PBS solutions per hole；2nd row is set as control group, often
Hole adds 100 μ L enzyme mark dilutions and 100 μ LPBS solution；3-8 rows are separately added into Hostathion titer successively per hole or sample carries
Take liquid gradient dilution liquid and 100 μ L enzyme mark dilutions.Phosphate Tween buffer PBST board-washings are used after being incubated at room temperature 1h five times.96
100-200 μ L substrate solutions are added per hole for hole elisa Plates, react 30-60min at room temperature.50-100 μ L1.25mol/L are added per hole
Sulfuric acid terminates reaction；96 hole elisa Plates 1-8 row absorbance value A are read with microplate reader, calculate separately inhibiting rate；
Inhibiting rate value of the various concentration Hostathion to antigen-antibody binding reaction is calculated according to the following formula：
IC%=[1- (ASample-ABlank)÷(AControl-ABlank)]×100；
In formula：
Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；
AControl--- the negative control hole mean absorbance values；
ASample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；
ABlank--- the mean absorbance values of the blank control wells；
Using the logarithm of Hostathion standard specimen gradient dilution liquid concentration as abscissa, corresponding inhibiting rate percentage is vertical sit
Mark, draws standard curve respectively.
The group of 5 times of PBST solution is divided into：200mL, 10% Tween-20：5mL adds distilled water to be settled to 1L；By 5
It is PBST solution that times PBST distilled water, which dilutes 5 times,.
The substrate solution is formulated as：Substrate A：8.0g anhydrous sodium acetates, 3.0g beta-cyclodextrins and 140mg hydrogen peroxide
Urea adds distilled water to be settled to 1000mL, adjusts pH to 5.2,4 DEG C of preservations；Substrate B：40mg 3,3,5,5- tetramethyl benzidines are dissolved in
6mL dimethyl sulfoxide (DMSO)s, room temperature are kept in dark place；Using preceding 15min, 14.6mL substrate As and 0.45mL substrate Bs is taken to be mixed into substrate
Liquid.
5. weighing 2g samples to be tested, 10mL PBS solutions ultrasonic extraction is added after crushing 3 times, filters to obtain sample extracting solution.
By sample extracting solution replace step 4) described in Hostathion standard specimen gradient dilution liquid, repeat step 4) operation, according to inhibiting rate by
Standard curve calculates the content of Hostathion in determinand.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but it will be understood by those of ordinary skill in the art that：Its
It still can be with technical scheme described in the above embodiments is modified, either to which part or all technical features
Carry out equivalent replacement；And these modifications or replacements, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. a kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion, which is characterized in that including：
Molecular imprinted polymer membrane ELISA Plate and Hostathion enzyme-labelled antigen；
It is described to be prepared by the following method to obtain：Using triazolone as template molecule, with function monomer, crosslinking agent, initiator lazy
Property solvent in carry out prepolymerization obtain prepolymerization liquid；The prepolymerization liquid is added in ELISA Plate, is eluted after vacuum drying polymerization
Template molecule to obtain the final product.
2. kit according to claim 1, which is characterized in that the function monomer is methacrylic acid, the crosslinking
Agent is trimethoxy oxypropyl trimethyl acrylate, and the template molecule：Function monomer：The molar ratio of crosslinking agent is 1:5~
7:2~4；
Preferably, the atent solvent is acetonitrile；It is furthermore preferred that the template molecule：Atent solvent=1mol：20~30ml；
Preferably, the initiator is azodiisobutyronitrile；It is furthermore preferred that the template molecule：Initiator=1mol：40~
60mg。
3. kit according to claim 2, which is characterized in that the condition of the vacuum drying polymerization is vacuum drying item
Lower 30 DEG C~50 DEG C polymerization 12h of part~for 24 hours.
4. kit according to claim 1, which is characterized in that the preparation method of the Hostathion enzyme-labelled antigen includes：
Hostathion haptens is synthesized into envelope antigen using mixed anhydride method, then by the envelope antigen and zymoprotein according to 18
~22:1 molar ratio is coupled；
Preferably, the zymoprotein is horseradish peroxidase.
5. kit according to claim 4, which is characterized in that described to close Hostathion haptens using mixed anhydride method
It is specifically included at the step of envelope antigen：By Hostathion haptens and n,N-Dimethylformamide with 0.2mmol~0.3mmol：
The ratio of 1ml is mixed to get mixed liquor, then be added into the mixed liquor positive tri-n-butylamine and ethyl chloroformate reaction 50min~
70min, then gained reaction solution is mixed with the CBS buffer solutions dissolved with OVA, after dialysis to obtain the final product.
6. according to Claims 1 to 5 any one of them kit, which is characterized in that the kit further includes buffer solution；It is excellent
Choosing, the buffer solution is PBS solution.
7. according to Claims 1 to 5 any one of them kit, which is characterized in that the kit further includes substrate solution；
The substrate solution is configured by substrate A and substrate B；
The ingredient of the substrate A includes：The anhydrous sodium acetate of 8.0g/L~8.4g/L, the beta-cyclodextrin of 2.0g/L~3.0g/L with
And the carbamide peroxide of 140mg/L~160mg/L, pH=4.8~5.2；
The ingredient of the substrate B includes that ratio is 40mg~60mg:The 3,3,5,5- tetramethyl benzidines of 4ml~6ml:Dimethyl
Sulfoxide.
8. a kind of carrying out the bionical MBP enzyme linked immuno-adsorbent assay of Hostathion using claim 1~7 any one of them kit
Method, which is characterized in that including：
Blank control wells, negative control hole and detection hole are set on the molecular imprinted polymer membrane ELISA Plate；
Hostathion enzyme-labelled antigen dilution and Hostathion titer or sample extracting solution are added in the detection hole, and different
The Hostathion titer or sample extracting solution being added in hole are by gradient dilution；
Hostathion enzyme-labelled antigen dilution and buffer solution are added in the negative control hole；
Wherein, the Hostathion enzyme-labelled antigen dilution is diluted to obtain by Hostathion enzyme-labelled antigen standard solution with buffer solution；
The buffer solution is added in the blank control wells；
Each hole is incubated at room temperature 50min~70min after being added in a manner described, substrate is separately added into each hole after board-washing
Liquid terminates reaction after being incubated at room temperature 30min~60min；Microplate reader, which reads each hole absorbance and calculates according to the following formula, to be inhibited
Rate：
IC%=[1- (ASample-ABlank)÷(AControl-ABlank)]×100；
In formula：
Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；
AControl--- the negative control hole mean absorbance values；
ASample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；
ABlank--- the mean absorbance values of the blank control wells；
Using the logarithm of Hostathion titer gradient dilution liquid or sample extracting solution gradient dilution liquid concentration as abscissa, accordingly
Inhibiting rate percentage is ordinate, draws standard curve respectively；
The corresponding absorbance value of the sample extracting solution is substituted into the standard curve to get the sample extracting solution concentration.
9. according to the method described in claim 8, it is characterized in that, the preparation method of the sample extracting solution includes：
Buffer solution ultrasonic extraction is added after sample to be tested is crushed, filters to obtain the final product.
10. according to the method described in claim 9, it is characterized in that, the ratio of the sample to be tested and the buffer solution is 1g
~2g：5ml~10ml.
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