CN104833781B - Magnetic molecular imprinting bionic ELISA (enzyme-linked immuno sorbent assay) detecting method of malachite green - Google Patents
Magnetic molecular imprinting bionic ELISA (enzyme-linked immuno sorbent assay) detecting method of malachite green Download PDFInfo
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Abstract
The invention discloses a magnetic molecular imprinting bionic ELISA (enzyme-linked immuno sorbent assay) detecting method. The magnetic molecular imprinting bionic ELISA detecting method includes steps of preparing magnetic molecular imprinted polymer and setting up a direct-competition ELISA method. By the detecting method, uniform-particle MG-MMIPs (malachite green-magnetic molecular imprinted polymers) are prepared by an emulsion polymerization method, adsorption performance is investigated, the MG-MMIPs is used as bionic antibody, and the directly-competitive ELISA detecting method is established by means of competitive adsorption to MG antigen and enzyme labeling MG antigen. Under the optimum reaction conditions, the standard curve sensitivity is 20.14 ugL-1, and the minimum detection limit is 0.12 ugL-1 and is lower than detection limit of 2 ppb (ng/g) of a MG rapid-detection card. Meanwhile, the method has high selectivity for MG, and cross reaction rates for two structural analogues (methyl violet and brilliant green) of the MG are 7.4% and 3.9% respectively.
Description
Technical field
The present invention relates to one kind is in a big way, specifically a kind of aquatic products malachite green oxalate it is magnetic molecularly imprinted bionical
ELISA detection method.
Background technology
Malachite green oxalate (Malachite Green, MG) is a kind of triphenylmethane material, is also called salt matrix green, alkaline
Peacock green, peacock green, Viride Nitenses, Chinese green or aniline green, are the bottle green crystalline solid with metallic luster.Malachite green oxalate
Once there are application, such as leather industry, food dyeing, ceramics, dyeing industry etc. in many industries.As MG is for aquatic animal
The epidemic prevention and treatment of disease can play good curative effect, start just to be widely used in aquaculture from 1936.Malachite
It is green also to be used as antibacterial, for preventing mycete from growing in fish roe and the secondary pollution of funguses has preferable effect.But,
Malachite green oxalate haves the shortcomings that high poison, high residue and height be carcinogenic, mutation.China has been set to aquatic biological in May, 2002
Violated medication, but it is cheap due to which, and also bactericidal effect is significantly, still has illegal trade company also using at present.As which adds
Dosage seldom can just reach good bactericidal effect, bring certain difficulty to the MG in detection aquatic products.Therefore, set up MG
The detection method of residual is very urgent and has important practical significance.
The detection method of MG mainly has high performance liquid chromatography, Liquid Chromatography-Mass Spectrometry and gas phase color at present
Spectrum-MS.These methods all have reliable results, sensitivity in the residual for determining and analyzing aquatic products Malachite Green
High, the reproducible advantage of high, selectivity.But all there is very complicated, time-consuming etc. in sample pretreatment process in these methods
Deficiency, and apparatus expensive, testing cost are high；Not only need to consume substantial amounts of solvent and substantial amounts of time, be also easy to cause two
Secondary pollution, affects the accuracy of testing result.Therefore, develop a kind of detection method of MG rapidly and efficiently to controlling Aquatic product quality
Amount safety is significant.
Molecularly imprinted polymer has the distinguishing feature of structure effect precordainment, specific recognition and broad applicability, and enzyme-linked
Immunization detection is quick, sensitive.The two is combined, the molecularly imprinted polymer using synthesis not only has as bionic antibody
There is selection specificity, and can reuse.
The content of the invention
It is an object of the invention to provide a kind of magnetic molecularly imprinted bionical ELISA detection sides of aquatic products malachite green oxalate
Method, to solve the problems, such as to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme：
A kind of magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate, comprises the following steps that：
(1)Fe3O4Synthesis：0.30～0.36g Polyethylene Glycol is weighed, 7～9mL ultra-pure waters are added, ultrasound is until poly- second two
Alcohol is completely dissolved, and weighs 2.00～2.06g FeCl3Ultrasonic dissolution in polyglycol solution is added to, then weighs 1.4～1.6g
FeCl2It is added in above-mentioned solution to being completely dissolved, adds 0.4～0.6mL0.1mol L immediately-1Dilute hydrochloric acid, letting nitrogen in and deoxidizing 12
～18min, is rapidly added 55～65mL ammonia under 60 DEG C of water-baths while stirring, turns speed to 1500r min-1, 1h is reacted, magnetic is used
Ferrum is adsorbed, and with ultrapure water elution 4～6 times, is saved backup after vacuum lyophilization；
(2) preparation of pre-polymer solution：By 0.08～0.12g Fe3O4Add 0.8～1.2mL Oleic acid in and use Glass rod
Stir, add 8～12mL 0.05mol L-1MG chloroformic solutions and 43～170 μ L function monomers, in 400r min-1Turn
Speed 25～35min of lower stirring, stands 1.5～2.5h and forms A liquid at 4 DEG C；
(3) preparation of pre-emulsification solution：Span-80 and tween-80 each 0.4～0.6mL is added into 22～28mL ultra-pure waters
In, 475～950 μ L cross-linking agent are added, in 400r min-125～35min of lower stirring forms B liquid；
(4) preparation of MG magnetic molecularly imprinted polymers：In 400r min-1Under rotating speed stirring, A liquid is slowly added into into B
In liquid, temperature is raised to 60 DEG C by nitrogen 18～22min of deoxygenation, add 0.08～1.2g azodiisobutyronitriles cause polymerization 16～
20h, reacts after terminating with 22～28mL methanol breakdowns of emulsion, obtains thick malachite green oxalate magnetic molecularly imprinted polymer, product after sucking filtration
It is 8～10 with volume ratio:1 methanol-acetic acid mixed liquor surname extraction is extremely washed out without malachite green oxalate template, during again with methanol is washed till
Property, finally methanol is washed away with ultra-pure water, it is standby after vacuum lyophilization；
(5) haptenic synthesis：By 850～950mg 4- formylbenzoates, 2.2～2.6mL N, accelerine
ZnCl anhydrous with 2.2～2.6g2It is dissolved in 60mL dehydrated alcohol, under nitrogen protection, is heated to reflux 22～26h, room temperature cooling
Afterwards, 28～32mL methanol, Deca ammonia is added to be rinsed with water after filtration, be dried with KOH in vacuum and obtained to generation is precipitated
Carboxylated concealed malachite green；Chloroform dissolves carboxylated concealed malachite green, adds chloranil, glacial acetic acid 25
1～2h of stirring reaction at DEG C, the isopyknic chloroform of product Jing-tetrachloromethane are washed 2～3 times, and vacuum drying can be obtained
Obtain the carboxylated malachite green oxalate powder of hapten；
(6) synthesis of enzyme-labelled antigen：1.8～2.2mg hapten carboxylated malachite green oxalate CMG powder is dissolved in into 0.8～
1.2mL 20%N, dinethylformamide solution add 1.8～2.2mg mL in magnetic agitation-132～24 μ L of EDC, adjust
PH 4.5～5.0, after 18～22min of reaction, is rapidly added 7.0～8.0mg horseradish peroxidase HRP, adjust pH to 7.8～
7.2,2.5～3.5h of room temperature reaction obtain CMG-HRP conjugates, 20 DEG C of freezen protectives；
(7) detect：Take 18～22mg magnetic molecularly imprinted polymers to be placed in 10mL centrifuge tubes, add concentration be 0.1～
10000μgL-1MG-PBS 2.8～3.2mL of buffer solution, immediately after add dilution factor be 1：1400～1600 enzyme mark resists
2.8～3.2mL of original solution, be not added with MG solution centrifuge tube be matched group, ice bath vibration absorption 1.5～2.5h；Abandon upper solution,
Often pipe adds 140～160 μ L substrate nitrite ions, and after 25～35min of colour developing, often pipe adds 45～55 μ L terminate liquids, immediately in enzyme mark
Reading on instrument, calculates suppression ratio of the MG to antigen-antibody binding reaction under each concentration.
As further scheme of the invention：Function monomer in step (2) is α-methacrylic acid, acrylamide
Or acrylic acid.
As further scheme of the invention：Cross-linking agent in step (3) be ethylene glycol dimethacrylate, two
Ethenylbenzene or trimethylol-propane trimethacrylate.
As further scheme of the invention：Substrate nitrite ion in step (7) is o-phenylenediamine.
As further scheme of the invention：Terminate liquid in step (7) is 2mol L-1Sulphuric acid.
Compared with prior art, the invention has the beneficial effects as follows：Magnetic molecularly imprinted polymer of the present invention has special knowledge
Other property and the distinguishing feature for making solid-liquid sharp separation, and euzymelinked immunosorbent assay (ELISA) detects the characteristics of having quick and sensitive, and the two is tied
Altogether, using the molecularly imprinted polymer of synthesis as bionic antibody, not only with selection specificity, and profit can be repeated
With.
Description of the drawings
Fig. 1 is magnetic molecularly imprinted polymer electron-microscope scanning figure in the present invention；
Fig. 2 is magnetic molecularly imprinted polymer FTIR spectrum figure in the present invention；
It is magnetic molecularly imprinted polymer hysteresis curve during Fig. 3 is of the invention；
Fig. 4 and Fig. 5 is magnetic molecularly imprinted polymer thermal multigraph in the present invention；
Fig. 6 is enzyme-labelled antigen ultraviolet spectrogram in the present invention；
Fig. 7 is hapten CMG infrared spectrograms in the present invention；
Fig. 8 is MG direct competive ELISA standard curves in the present invention；
Fig. 9 is concordance of the ELISA and HPLC methods to MG measurement results in sample.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
The embodiment of the present invention
A kind of magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate, comprises the following steps that：
(1)Fe3O4Synthesis：0.33g Polyethylene Glycol is weighed, 8mL ultra-pure waters are added, ultrasound is until Polyethylene Glycol is completely molten
Solution, weighs 2.03g FeCl3Ultrasonic dissolution in polyglycol solution is added to, then weighs 1.5gFeCl2It is added in above-mentioned solution
To being completely dissolved, 0.5mL 0.1mol L are added immediately-1Dilute hydrochloric acid, letting nitrogen in and deoxidizing 15min are rapid while stirring under 60 DEG C of water-baths
60mL ammonia is added, and speed is turned to 1500r min-1, 1h is reacted, is adsorbed with Magnet, and with ultrapure water elution 5 times, vacuum
Save backup after lyophilization；
(2) preparation of pre-polymer solution：By 0.1g Fe3O4Stir in adding 1mL Oleic acid and with Glass rod, add
10mL 0.05mol L-1MG chloroformic solutions and 110 μ L function monomers, in 400r min-1Rotating speed under stir 30min, at 4 DEG C
Stand 2h and form A liquid；
(3) preparation of pre-emulsification solution：Span-80 and tween-80 each 0.5mL is added in 25mL ultra-pure waters, is added
700 μ L cross-linking agent, in 400r min-1Lower stirring 30min forms B liquid；
(4) preparation of MG magnetic molecularly imprinted polymers：In 400r min-1Under rotating speed stirring, A liquid is slowly added into into B
In liquid, temperature is raised to 60 DEG C by nitrogen deoxygenation 20min, adds 0.1g azodiisobutyronitriles to cause polymerization 18h, after reaction terminates
With 25mL methanol breakdowns of emulsion, thick malachite green oxalate magnetic molecularly imprinted polymer after sucking filtration, is obtained, product volume ratio is 9:1 first
To washing out without malachite green oxalate template, again with methanol is washed till neutrality, finally washes away first with ultra-pure water alcohol-acetic acid mixture surname extraction
Alcohol, it is standby after vacuum lyophilization；
(5) haptenic synthesis：By 900mg 4- formylbenzoates, 2.4mL N, accelerine and 2.4g are anhydrous
ZnCl2It is dissolved in 60mL dehydrated alcohol, is heated to reflux 24h under nitrogen protection, after room temperature cooling, adds 30mL methanol, Deca
Ammonia is rinsed with water after filtration, is dried with KOH and obtains carboxylated concealed malachite green in vacuum to generation is precipitated；Three
Chloromethanes dissolve carboxylated concealed malachite green, and stirring reaction 1.5h at addition chloranil, 25 DEG C of glacial acetic acid, reaction are produced
Isopyknic chloroform-the tetrachloromethanes of thing Jing are washed twice, and vacuum drying can obtain the carboxylated malachite green oxalate powder of hapten；
(6) synthesis of enzyme-labelled antigen：2mg hapten carboxylated malachite green oxalate CMG powder is dissolved in into 1mL 20%N, N- bis-
Methylformamide solution, adds 2mg mL in magnetic agitation-133 μ L of EDC, adjust pH4.5～5.0, after reaction 20min, rapidly
7.5mg horseradish peroxidase HRP are added, adjusts pH to 7.0, room temperature reaction 3h to obtain CMG-HRP conjugates, 20 DEG C of freezings are protected
Deposit；
(7) detect：Take 20mg magnetic molecularly imprinted polymers to be placed in 10mL centrifuge tubes, add concentration to be 0.1～10000
μgL-1MG-PBS buffer solution 3mL, immediately after add dilution factor be 1：1500 enzyme-labelled antigen solution 3mL, is not added with MG molten
The centrifuge tube of liquid be matched group, ice bath vibration absorption 2h；Upper solution is abandoned, often pipe adds 150 μ L substrate nitrite ion o-phenylenediamines,
After colour developing 30min, often pipe adds 50 μ L terminate liquid 2mol L-1Sulphuric acid, the reading in microplate reader, calculates under each concentration immediately
Suppression ratio of the MG to antigen-antibody binding reaction,
Suppression ratio (B/B0)=sample absorbance (B)/feminine gender absorbance (B0)；
Draw MG direct competive ELISA standard curves as shown in Figure 5.
Preferably, the function monomer in step (2) is α-methacrylic acid, acrylamide or acrylic acid.
Cross-linking agent in preferred step (3) is ethylene glycol dimethacrylate, divinylbenzene or trihydroxy methyl
Propane trimethyl acrylic ester.
As shown in figure 1, the electron-microscope scanning of the magnetic molecularly imprinted polymer (left side) prepared and non-imprinted polymer (right side)
Figure, the pattern rule of polymer, uniform particle sizes, in 600nm or so.
Figure it is seen that the infrared spectrogram of MIP and NIP is basically identical, this shows that template molecule is almost washed completely
It is de-.In polymer, main functional group can be embodied by corresponding vibration peak, wherein 2959cm-1Place is methyl (- CH3)
Stretching vibration peak, 1737cm-1Locate the stretching vibration peak for polymer carboxyl (C=O), 1641cm-1Place is double in polymer
The stretching vibration peak of key (C=C), 584cm-1Place is the stretching vibration peak of Fe-O keys.
From the figure 3, it may be seen that magnetic molecularly imprinted polymer has good superparamagnetism, saturation magnetization intensity is 54.1emu/
g.It can thus be appreciated that the MMIPs for preparing can be separated from sample rapidly under additional the action of a magnetic field.
From Fig. 4 and Fig. 5, when temperature is less than 265 DEG C, NIP is in a relatively steady state, almost no matter
The loss of amount；After temperature is increased to 265 DEG C, the quality of NIP starts to reduce in large quantities, when temperature reaches 405 DEG C, NIP's
Mass lost amount reaches 80%；MIP is very stable when temperature is less than 268 DEG C, does not almost have the loss of quality；When temperature is higher than
When 268 DEG C, mass loss is begun with, when temperature reaches 412 DEG C, Mass lost amount reaches 70%, illustrate magnetic molecularly imprinted poly-
Compound has good heat stability.
It will be appreciated from fig. 6 that CMG has characteristic absorption peak at 629,429,320,251nm, HRP has at 403 and 268nm
Characteristic absorption peak, and enzyme-labelled antigen has characteristic absorption peak at 628,291,226nm, while some features of CMG and HRP are inhaled
Receive peak to disappear, illustrate that enzyme-labelled antigen is coupled successfully.
As shown in fig. 7, functional group main in carboxylated malachite green oxalate can be embodied by corresponding vibration peak, its
Middle 3462cm-1Place is the stretching vibration peak of O-H, 1559cm-1And 1449cm-1Locate the stretching vibration peak for C=C on phenyl ring,
955cm-1、691cm-1Place is the stretching vibration of phenyl ring.
The specificity verification of set up ELISA method：Select malachite green oxalate and its analog crystal violet, viride nitens conduct
Analyte, sets up dcELISA standard curves, respectively obtains corresponding IC50 values, calculates cross reacting rate (CR), such as table 1
As a result show, prepared magnetic molecularly imprinted polymer has higher selectivity to malachite green oxalate.
The IC of 1 MG of table and the like50Value and cross reacting rate
The addition recovery experiment of actual sample：By 5 μ g L-1、10μg L-1、50μg L-1Malachite green oxalate standard solution adds
To in Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) and shrimp sample, then using the dcELISA determination sample response rate set up, experimental result table as shown in table 2
Bright, the method set up can be applied in actual sample detection, and Detection results are preferable.
Addition recovery experiment of 2 ELISA method of table to MG in sample
The Accuracy Verification of ELISA method：By 5 μ g L-1、10μg L-1、50μg L-1Malachite green oxalate standard solution is added to
In Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) and shrimp sample, then it is measured using high performance liquid chromatography direct injected, the measure as shown in table 3 and Fig. 8
As a result show, the ELISA method set up and HPLC methods are to adding the Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) of variable concentrations MG and the measurement result of shrimp sample
With reasonable concordance, the bionical enzyme immunoassay detection method set up has higher accuracy and practicality.
Measurement result is reclaimed in the addition of 3 ELISA method of table and HPLC methods to MG in sample
Claims (5)
1. a kind of magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate, it is characterised in that concrete steps are such as
Under：
(1)Fe3O4Synthesis：0.30～0.36g Polyethylene Glycol is weighed, 7～9mL ultra-pure waters are added, ultrasound is until Polyethylene Glycol is complete
CL, weighs 2.00～2.06g FeCl3Ultrasonic dissolution in polyglycol solution is added to, then weighs 1.4～1.6g FeCl2
It is added in above-mentioned solution to being completely dissolved, adds 0.4～0.6mL 0.1mol L immediately-1Dilute hydrochloric acid, letting nitrogen in and deoxidizing 12～
18min, is rapidly added 55～65mL ammonia under 60 DEG C of water-baths while stirring, turns speed to 1500r min-1, 1h is reacted, Magnet is used
Adsorbed, and with ultrapure water elution 4～6 times, saved backup after vacuum lyophilization；
(2) preparation of pre-polymer solution：By 0.08～0.12g Fe3O4Stir in adding 0.8～1.2mL Oleic acid and with Glass rod
Uniformly, 8～12mL 0.05mol L are added-1MG chloroformic solutions and 43～170 μ L function monomers, in 400r min-1Rotating speed under
25～35min of stirring, stands 1.5～2.5h and forms A liquid at 4 DEG C；
(3) preparation of pre-emulsification solution：Span-80 and tween-80 each 0.4～0.6mL is added in 22～28mL ultra-pure waters,
475～950 μ L cross-linking agent are added, in 400r min-125～35min of lower stirring forms B liquid；
(4) preparation of MG magnetic molecularly imprinted polymers：In 400r min-1Under rotating speed stirring, A liquid is slowly added in B liquid,
Temperature is raised to 60 DEG C by nitrogen 18～22min of deoxygenation, adds 0.08～1.2g azodiisobutyronitriles to cause 16～20h of polymerization, instead
With 22～28mL methanol breakdowns of emulsion after should terminating, thick malachite green oxalate magnetic molecularly imprinted polymer, product volume after sucking filtration, is obtained
Than for 8～10:To washing out without malachite green oxalate template, again with methanol is washed till neutrality to 1 methanol-acetic acid mixed liquor surname extraction, most
Methanol is washed away with ultra-pure water afterwards, it is standby after vacuum lyophilization；
(5) haptenic synthesis：By 850～950mg 4- formylbenzoates, 2.2～2.6mL N, accelerine and
The anhydrous ZnCl of 2.2～2.6g2It is dissolved in 60mL dehydrated alcohol, under nitrogen protection, is heated to reflux 22～26h, after room temperature cooling,
28～32mL methanol, Deca ammonia is added to be rinsed with water after filtration, be dried with KOH in vacuum and obtain carboxylic to generation is precipitated
The concealed malachite green of base；Chloroform dissolves carboxylated concealed malachite green, adds chloranil, 25 DEG C of glacial acetic acid
1～2h of lower stirring reaction, the isopyknic chloroform of product Jing-tetrachloromethane are washed 2～3 times, and vacuum drying can be obtained
The carboxylated malachite green oxalate powder of hapten；
(6) synthesis of enzyme-labelled antigen：1.8～2.2mg hapten carboxylated malachite green oxalate CMG powder is dissolved in into 0.8～1.2mL
20%N, dinethylformamide solution add 1.8～2.2mg mL in magnetic agitation-132～24 μ L of EDC, adjust pH 4.5
～5.0, after 18～22min of reaction, 7.0～8.0mg horseradish peroxidase HRP are rapidly added, adjust pH to 7.8～7.2, room temperature
2.5～3.5h of reaction obtains CMG-HRP conjugates, 20 DEG C of freezen protectives；
(7) detect：Take 18～22mg magnetic molecularly imprinted polymers to be placed in 10mL centrifuge tubes, add concentration to be 0.1～10000
μgL-1MG-PBS 2.8～3.2mL of buffer solution, immediately after add dilution factor be 1：1400～1600 enzyme-labelled antigen solution
2.8～3.2mL, be not added with MG solution centrifuge tube be matched group, ice bath vibration absorption 1.5～2.5h；Upper solution is abandoned, often pipe adds
Enter 140～160 μ L substrate nitrite ions, often pipe adds 45～55 μ L terminate liquids after 25～35min of colour developing, reads immediately in microplate reader
Number, calculates suppression ratio of the MG to antigen-antibody binding reaction under each concentration.
2. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature
It is that the function monomer in step (2) is α-methacrylic acid, acrylamide or acrylic acid.
3. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature
It is that the cross-linking agent in step (3) is ethylene glycol dimethacrylate, divinylbenzene or trimethylol propane trimethyl
Acrylate.
4. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature
It is that the substrate nitrite ion in step (7) is o-phenylenediamine.
5. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature
It is that the terminate liquid in step (7) is 2mol L-1Sulphuric acid.
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| CN105628761B (en) * | 2015-06-06 | 2019-05-03 | 南华大学 | Malachite green voltammetric sensor based on print identification and the preparation method and application thereof |
| CN105242038A (en) * | 2015-11-13 | 2016-01-13 | 集美大学 | Aquatic product malachite green molecularly imprinted membrane bionic ELISA quick detection method |
| CN106582567B (en) * | 2016-11-30 | 2019-07-26 | 河南科技大学 | The preparation method and applications of organic Arsiness magnetic molecularly imprinted polymer |
| CN108084341B (en) * | 2017-11-03 | 2020-06-02 | 仲恺农业工程学院 | Synthesis method and application of crystal violet molecularly imprinted microspheres |
| CN109507126B (en) * | 2018-12-07 | 2021-01-01 | 集美大学 | Method for detecting malachite green in aquatic product |
| CN112379098B (en) * | 2020-11-05 | 2022-04-15 | 集美大学 | ELISA detection method for mimic enzyme-labeled antibody of histamine content in aquatic product |
| CN113896651B (en) * | 2021-12-06 | 2022-03-08 | 信达安检测技术(天津)有限公司 | Synthesis method of dominant malachite green hapten and application of hapten |
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| US20060177882A1 (en) * | 2005-02-04 | 2006-08-10 | Samad Talebpour | Immunoassays with enhanced selectivity |
| CN101226194B (en) * | 2008-01-18 | 2011-11-16 | 华南农业大学 | Malachite green vestigial ELISA detection kit and usage method thereof |
| CN101482559A (en) * | 2009-02-12 | 2009-07-15 | 四川大学 | ELISA adsorption analysis method for measuring gross amount of malachite green and colorless malachite green in water sample and aquatic products |
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| CN102353775B (en) * | 2011-06-13 | 2014-04-02 | 清华大学深圳研究生院 | Immunochromatographic test strip for detecting malachite green (MG) and preparation process thereof |
| CN103105386B (en) * | 2013-01-29 | 2015-05-20 | 福州大学 | Method for detecting malachite green in water body and aquatic products |
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| CN103558203B (en) * | 2013-11-22 | 2015-07-01 | 中国农业科学院农业质量标准与检测技术研究所 | Magnetic molecularly imprinted polymer-fluorescence analysis method |
| CN103728448A (en) * | 2013-12-31 | 2014-04-16 | 上海海洋大学 | Preparation method of horse radish peroxidase labeled leuco-malachite green immunogen |
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