CN104655848A - Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit - Google Patents

Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit Download PDF

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CN104655848A
CN104655848A CN201510020462.4A CN201510020462A CN104655848A CN 104655848 A CN104655848 A CN 104655848A CN 201510020462 A CN201510020462 A CN 201510020462A CN 104655848 A CN104655848 A CN 104655848A
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ractopamine
ptnps
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CN104655848B (en
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潘道东
陈淑贤
孙杨赢
曹锦轩
曾小群
吴振
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Ningbo University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/9413—Dopamine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

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Abstract

The invention discloses an enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as a preparation method and the application of the detection kit. The detection kit is characterized in that Fe3O4@beta-CD is taken as a capture probe, and a compound of colloidal platinum particles and polymerase chelate PV is taken as a signal probe; the preparation method of the detection kit comprises the steps of preparing Fe3O4-NH2; preparing the capture probe Fe3O4@beta-CD; preparing PtNPs; preparing a PtNPs/ PV signal label; and preparing an immune complex. The application comprises the following steps: feeding 50mu L of liquid A and 50mu L of liquid B of diaminobenzidine (DAB) into the obtained immune complex; carrying out light-avoiding incubation for 15-30 minutes; measuring the absorbance of the obtained DAB developed product by a microplate reader; drawing a standard curve according to the relation between corresponding light absorption values and concentrations of the ractopamine; and determining the concentration of the ractopamine in a sample to be tested according to the standard curve. The detection kit has the advantages of being high in sensitivity, selectivity and accuracy as well as rapid in detection speed.

Description

Detect enzyme-linked immunologic detecting kit of Ractopamine and its preparation method and application
Technical field
The present invention relates to Ractopamine detection technique, especially relate to a kind of based on Fe 3o 4@β-CD as capture probe, PtNPs/PV as signal probe for enzyme-linked immunologic detecting kit detecting Ractopamine and its preparation method and application.
Background technology
β 2receptor stimulating agent (β-adrenergic agonists) because of can relieving asthma symptoms, thus be often used as veterinary drug; But be also often added in feed because it can increase muscle, minimizing lipopexia etc.Ractopamine (Ractopamine, RAC) is as β 2a member of receptor stimulating agent is a kind of Ke Lunbaan of Prof. Du Yucang, is just used on some pig farms by as the novel clenbuterol hydrochloride of one.Provide against Ractopamine in " forbidding the types of drugs catalogue used in feed and animal drinking water " that the Ministry of Agriculture of China, the Ministry of Public Health, state food drug surveilance office combine issue to use in feed as growth promoter.Therefore, a kind of quick, high-sensitive Ractopamine trace residue detection method is needed badly as the important means ensureing food security.
At present, a large amount of detection meanss after deliberation and for the detection of Ractopamine, wherein comparatively accurately reliably as gas chromatography (GC), liquid chromatography (LC) and they and mass spectrometry (GC-MS, LC-MS).But all there is the problems such as instrumentation complexity, expensive equipment, length consuming time in them, these problems make them be difficult to on-site quick screening.Although Rapid detection test strip facilitates but only can accomplish qualitative, can not be quantitative.Meanwhile, traditional Ractopamine ELISA detection kit generally uses competition law, although effective and rapid, but still there is the shortcomings such as detectability is too high, false positive.Also do not disclose any about utilizing Fe at present both at home and abroad 3o 4@β-CD reports for the correlative study detecting the immune colourimetry of Ractopamine as enzyme labelled antibody as capture probe, PtNPs/PV compound.
Summary of the invention
Technical matters to be solved by this invention is to provide that a kind of detection speed is fast, accuracy and highly sensitive, high specificity and being easy to operates for enzyme-linked immunologic detecting kit detecting Ractopamine and its preparation method and application.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of enzyme-linked immunologic detecting kit for detecting Ractopamine, comprises ELISA Plate and signal probe, and described ELISA Plate is fixed with Ractopamine antigen and capture probe Fe 3o 4@β-CD, described signal probe is the compound of the PtNPs/PV of a large amount of PtNPs of load, and standard items are Ractopamine standard items, and antibody working fluid is anti-Anti-ractopamine antibody monoclonal antibody, nitrite ion A, nitrite ion B are developer DAB, and stop buffer is 2M HCl or 2M H 2sO 4, washing lotion is PBST damping fluid.
For detecting a preparation method for the enzyme-linked immunologic detecting kit of Ractopamine, concrete steps are as follows:
(1) Fe 3o 4-NH 2preparation
By anhydrous sodium acetate, hexane diamine (1,6-hexanediamine) and FeCl 36 H 2join in ethylene glycol after O mixing, stir under 42-60 DEG C of condition and obtain source of iron solution, source of iron solution is transferred in muffle furnace, react 6-8h under 195-205 DEG C of condition after, be separated with magnet, use water and alcohol flushing 2-3 time more respectively, remove the solvent of non-complete reaction, obtain the Fe that particle diameter is 15-25 nm 3o 4-NH 2magnetic nanoparticle;
(2) Fe 3o 4the preparation of@β-CD
N-Hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) are added to containing carboxymethylation beta-schardinger dextrin-(carboxymethyl-β-CD, CM-β-CD) mass concentration is in the PBS solution (pH=7.4) of 1wt%, at room temperature stir after spending the night (to activate the ethyloic on beta-schardinger dextrin-), then add the Fe that step (1) prepares 3o 4-NH 2magnetic nanoparticle, after stirring 5-7h continuously, is separated with magnet, more repeatedly cleans with deionized water, to remove unreacted chemical substance, and the Fe obtained 3o 4@β-CD;
(3) preparation of PtNPs
The platinum acid chloride solution of 2ml 1wt% is joined after gently stirring 4-6min in beaker, add rapidly the sodium borohydride solution reduction of 5ml 10mmol/L, vigorous stirring under room temperature, by removing impurity in the centrifugal 15-25min of 10000-12000r/min after reaction terminates, be settled to 2ml after washing at least 3 times and obtain PtNPs solution, save backup under 4 DEG C of conditions; Wherein beaker soaks through chloroazotic acid;
(4) preparation of PtNPs/PV complex signal label
PtNPs solution step (3) prepared is gentle agitation 5-10min under 0-4 DEG C (when adding to make PV enzyme, temperature is unlikely to too high and makes enzyme deactivation), slowly drip equal-volume PV enzyme reagent (PowerVision under agitation, purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), stir the bovine serum albumen solution adding 1wt% after spending the night, keep 2-6 hour (closing key mapping unnecessary on PtNPs) in low temperature 0-4 DEG C, namely obtain PtNPs/PV complex solution, save backup under 4 DEG C of conditions;
(5) preparation of enzyme linked immunological kit immune complex
5 mgFe are added in every hole of ELISA Plate 3o 4standard items (0.03,0.1,0.3,0.9,2.7, the 8.1 ng mL of the Ractopamine of@β-CD and 50 μ L serial dilutions -1), the anti-Anti-ractopamine antibody solution of 50 μ L, mixes rear 37 DEG C of lucifuges and hatches 30min, PBST damping fluid (the 0.01 mol L containing 0.05 v/v % tween -1the PBS solution of pH 7.2-7.4) wash plate 3 times, magnet is separated, and then every hole adds 80 μ L PtNPs/PV complex solutions, mix rear 37 DEG C of lucifuges and hatch 30min, PBST damping fluid washes plate 3 times, and magnet is separated, and namely obtains kit for testing lecdopamine ELISA; Wherein the dilution of Ractopamine standard items is 0.01 mol L -1the PBS solution (PBS phosphate buffer is purchased from Beijing Suo Laibao Science and Technology Ltd.: 0.01M pH 7.2-7.4) of pH 7.2-7.4.
Anhydrous sodium acetate, hexane diamine (1,6-hexanediamine), FeCl in step (1) 36 H 2the blending ratio of O and ethylene glycol is 4g:13g:2g:30ml.
N-Hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, carboxymethylation beta-schardinger dextrin-and Fe in step (2) 3o 4-NH 2the mass ratio of magnetic nanoparticle is 1:1:2:1.
(DAB nitrite ion is the medicine inside Ractopamine detection kit to add diaminobenzidine in every hole of the kit for testing lecdopamine ELISA obtained in step (5), Ractopamine kit is purchased from Beijing Huaan Magnech Bio-Tech Co., Ltd., production code member: HE09003) A liquid and each 50 μ L of B liquid, lucifuge hatches 15-30min, and the DAB color product obtained measures absorbance by microplate reader.
The enzyme-linked immunologic detecting kit applying above-mentioned detection Ractopamine detects a method for Ractopamine concentration, and concrete steps are as follows: with Fe 3o 4@β-CD magnetic nanoparticle catches the Ractopamine (RAC) in solution as capture probe, adopt the antibody that the method for noncompetitive immunity makes the RAC specific bond on capture probe add, by wash plate and magnetic resolution remove free antigen-antibody bond and other educt only more than be fixed on antibody at the bottom of plate; Add colloidal platinum mark PV and antibody specific bond again, through hatching and washing plate, leave the immune complex of Ractopamine antigen, antibody, colloidal platinum mark PV composition, the amount of this immune complex increases with the increase of Ractopamine in sample, finally add DAB colour developing, carry out colorimetric analysis, with the concentration of Ractopamine in colorimetric analysis result determination testing sample.
Compared with prior art, the invention has the advantages that: the present invention makes public for the first time enzyme-linked immunologic detecting kit detecting Ractopamine and its preparation method and application, PowerVision(PV) reagent is a kind of polymerase chelate, containing antibody and a large amount of horseradish peroxidase (HRP), compared to traditional enzyme labelled antibody, this reagent adds the HRP enzyme that unit antibody marks, and is reached the object of signal amplification by the method increasing HRP enzyme content.Meanwhile, Pt colloids particle (PtNPs) has very strong catalytic effect to HRP catalytic substrate diaminobenzidine (Diaminobenzidine, DAB), and the addition of this catalytic effect and PtNPs is proportionate relationship.Further, PtNPs synthesis step is simple, particle stabilized, have very strong biocompatibility, can be combined with zymoprotein and can not affect its catalytic effect, can be used to load proteinase.
The present invention uses beta-schardinger dextrin-(β-cyclodextrins, β-CD) to catch the Ractopamine of trace in sample liquid as capture probe.Beta-schardinger dextrin-is as a kind of Subjective and Objective material, its inclusion technique medicine and Applications in Food Industry further extensive, its can phenol structure in selective adsorption sample liquid, and Ractopamine two end is symmetric phenol structure, thus can effectively enter beta-schardinger dextrin-inner chamber and realize antigen and fix.Because beta-schardinger dextrin-is a kind of soluble polysaccharide, in order to realize the object of sample separation, by (Fe on grafted by beta cyclodextrin to amination tri-iron tetroxide 3o 4-NH 2), utilize that externally-applied magnetic field reaches fast, simple separation.Fe 3o 4-NH 2particle diameter is less, within the scope of ultraviolet 230-1000nm, do not have characteristic absorption peak, does not affect the mensuration of colour developing result.
In sum, the present invention's enzyme-linked immunologic detecting kit detecting Ractopamine and its preparation method and application, utilizes Fe 3o 4@β-CD as capture probe, PtNPs/PV as the immune colorimetric determination Rct opamine residue of signal probe.Fe 3o 4@β-CD is as trace RAC in a large amount of enrichment solution of capture probe energy; PtNPs colloidal solid itself has obvious catalyzed coloration effect to chromogenic substrate DAB; The height that PV enzyme reagent is rich in horseradish peroxidase (HRP enzyme) as one simultaneously gathers multienzyme complex, and HRP a large amount of on it can effectively develop the color by catalysis DAB, and compared to traditional enzyme labelled antibody, PV has extremely strong enlarge-effect; In addition, PtNPs colloidal solid has very strong biocompatibility, can combine with PV but not affect the catalytic effect of PV.The present invention replaces the enzyme labelled antibody of conventional reagents box by PtNPs mark PV, co-catalysis substrate DAB produces color change, to reach dual amplification effect, thus realize reliable, quick, sensitive, the high specific detection of Ractopamine trace residue in food, thus in food, medical science etc., there is its huge using value.
Accompanying drawing explanation
Fig. 1 is the Fe of preparation 3o 4-NH 2scanning electron microscope (SEM) photograph;
Fig. 2 is the Fe of preparation 3o 4the scanning electron microscope (SEM) photograph of@β-CD;
Fig. 3 is the transmission electron microscope picture of the PtNPs of preparation;
Fig. 4 is the transmission electron microscope picture of the PtNPs/PV compound of preparation;
Fig. 5 is the phenogram of preparation PtNPs/PV compound; A: the ultraviolet-visible absorption spectroscopy of the PtNPs of preparation; B: the ultraviolet-visible absorption spectroscopy of polymerase compound PV; C: the ultraviolet-visible absorption spectroscopy of the PtNPs/PV compound of preparation;
Fig. 6 is based on the schematic diagram of PtNPs/PV as signal probe immunity colorimetric determination Ractopamine;
Fig. 7 is the canonical plotting that object Ractopamine light absorption value and Ractopamine concentration are set up.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
For detecting an enzyme linked immunological kit for Ractopamine, comprise ELISA Plate and signal probe, ELISA Plate is fixed with Ractopamine antigen and capture probe Fe 3o 4@β-CD, signal probe is the compound of the PtNPs/PV of a large amount of PtNPs of load, and standard items are Ractopamine standard items, and antibody working fluid is anti-Anti-ractopamine antibody monoclonal antibody, nitrite ion A, nitrite ion B are developer DAB, and stop buffer is 2M HCl or 2M H 2sO 4, washing lotion is PBST damping fluid.
This colourimetry is mainly by Fe 3o 4@β-CD magnetic nanoparticle catches the Ractopamine (RAC) in solution as capture probe, and the antibody adopting the method for noncompetitive immunity to make the RAC specific bond on capture probe to add, antibody on this antibody capable specific bond PtNPs/PV compound, through hatching and magnetic resolution, leave the immune complex of capture probe, Ractopamine antigen, antibody, PtNPs/PV composition.The amount of this immune complex increases with the increase of Ractopamine in sample, linear with the Ractopamine in sample.Finally add DAB colour developing, carry out colorimetric analysis.With the concentration of Ractopamine in colorimetric analysis result determination testing sample.
Specific embodiment two
For detecting a preparation method for the enzyme-linked immunologic detecting kit of Ractopamine, concrete steps are as follows:
(1) Fe 3o 4-NH 2preparation
By anhydrous sodium acetate, hexane diamine (1,6-hexanediamine) and FeCl 36 H 2join in ethylene glycol after O mixing, stir under 42-60 DEG C of condition and obtain source of iron solution, source of iron solution is transferred in muffle furnace, react 6-8h under 195-205 DEG C of condition after, be separated with magnet, use water and alcohol flushing 2-3 time more respectively, remove the solvent of non-complete reaction, obtain the Fe that particle diameter is 15-25 nm 3o 4-NH 2magnetic nanoparticle; Wherein anhydrous sodium acetate, hexane diamine (1,6-hexanediamine), FeCl 36 H 2o and be 4g:13g:2g:30ml by the blending ratio of ethylene glycol; Fig. 1 is the Fe of preparation 3o 4-NH 2scanning electron microscope (SEM) photograph; Prepared magnetic Fe 3o 4-NH 2the particle diameter of particle is about 17nm, and it is rough, and in a large amount of gullies shape, this structure makes this magnetic Fe 3o 4-NH 2there is great surface area for the load of β-CD, thus improve the adsorbability of capture probe;
(2) Fe 3o 4the preparation of@β-CD
N-Hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) are added to containing carboxymethylation beta-schardinger dextrin-(carboxymethyl-β-CD, CM-β-CD) mass concentration is in the PBS solution (pH=7.4) of 1wt%, at room temperature stir after spending the night (to activate the ethyloic on beta-schardinger dextrin-), then add the Fe that step (1) prepares 3o 4-NH 2magnetic nanoparticle, after stirring 5-7h continuously, is separated with magnet, more repeatedly cleans with deionized water, to remove unreacted chemical substance, and the Fe obtained 3o 4@β-CD; Wherein N-Hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, carboxymethylation beta-schardinger dextrin-and Fe 3o 4-NH 2the mass ratio of magnetic nanoparticle is 1:1:2:1; Fig. 2 is the Fe of preparation 3o 4the scanning electron microscope (SEM) photograph of@β-CD; Comparison diagram 4 can be found out, after combining β-CD, the particle diameter of this magnetic-particle increases, and this illustrates capture probe Fe 3o 4@β-CD successfully synthesizes;
(3) preparation of PtNPs
The platinum acid chloride solution of 2ml 1wt% is joined after gently stirring 4-6min in beaker, add rapidly the sodium borohydride solution reduction of 5ml 10mmol/L, vigorous stirring under room temperature, impurity is removed in the centrifugal 15-25min of 10000-12000r/min after reaction terminates, be settled to 2ml after washing at least 3 times and obtain PtNPs solution, save backup under 4 DEG C of conditions; Wherein beaker soaks through chloroazotic acid; Wherein beaker soaks through chloroazotic acid; Fig. 3 is the transmission electron microscope picture of the PtNPs of preparation, and prepared PtNPs particle presents more spherical, and its particle diameter is about 8 nm;
(4) preparation of PtNPs/PV complex signal label
PtNPs solution step (3) prepared is gentle agitation 5-10min under 0-4 DEG C (when adding to make PV enzyme, temperature is unlikely to too high and makes enzyme deactivation), slowly drip equal-volume PV enzyme reagent under agitation purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, model: PV-6000, specification: 3 ml, English name: Polymer Detection System For Immuno-Histological Staining, PowerVision is) in document, stir the bovine serum albumen solution adding 1wt% after spending the night, keep 2-6 hour (closing key mapping unnecessary on PtNPs) in low temperature 0-4 DEG C, namely PtNPs/PV complex solution is obtained, save backup under 4 DEG C of conditions, wherein PtNPs solution and the mixed volume of POWERVISION reagent are than being 1:1, Fig. 4 is the transmission electron microscope picture of the PtNPs/PV compound of preparation, and the PtNPs particle of comparison diagram 3 can be found out, in PtNPs/PV compound, PtNPs even particulate dispersion is on protein, and this illustrates that PtNPs is successfully attached on PV enzyme-antibody multimer chelate, Fig. 5 is the phenogram of preparation PtNPs/PV compound, a: the ultraviolet-visible absorption spectroscopy of the PtNPs of preparation, b: the ultraviolet-visible absorption spectroscopy of polymerase compound PV, c: the ultraviolet-visible absorption spectroscopy of the PtNPs/PV compound of preparation, simple colloidal platinum particle without obvious absorption peaks, only has certain absorbance at below 300nm within the scope of 230-1000nm, sees Fig. 5 a.Simple PV has absorption peak at 280nm, 376nm, 506nm place, as shown in Figure 5 c.Fig. 5 b is the signal probe after closing with BSA, and it has simple colloidal platinum particle and the characteristic absorption peak of PowerVision reagent simultaneously, and its larger absorbing proteins peak part derives from BSA.This illustrates that the PtNPs/PV compound that colloidal platinum and PowerVision reagent are combined into successfully synthesizes;
(5) preparation of enzyme linked immunological kit immune complex
5 mgFe are added in every hole of ELISA Plate 3o 4standard items (0.03,0.1,0.3,0.9,2.7, the 8.1 ng mL of the Ractopamine of@β-CD and 50 μ L serial dilutions -1), the anti-Anti-ractopamine antibody solution of 50 μ L, mixes rear 37 DEG C of lucifuges and hatches 30min, PBST damping fluid (the 0.01 mol L containing 0.05 v/v % tween -1the PBS solution of pH 7.2-7.4) wash plate 3 times, magnet is separated, and then every hole adds 80 μ L PtNPs/PV complex solutions, mix rear 37 DEG C of lucifuges and hatch 30min, PBST damping fluid washes plate 3 times, and magnet is separated, and namely obtains kit for testing lecdopamine ELISA; Wherein the dilution of Ractopamine standard items is 0.01 mol L -1the PBS solution (PBS phosphate buffer is purchased from Beijing Suo Laibao Science and Technology Ltd.) of pH 7.2-7.4.
In every hole of kit for testing lecdopamine ELISA, adding diaminobenzidine, (DAB nitrite ion is the medicine inside Ractopamine detection kit, Ractopamine kit is purchased from Beijing Huaan Magnech Bio-Tech Co., Ltd., production code member: HE09003) A liquid and each 50 μ L of B liquid, lucifuge hatches 15-30min, and the DAB color product obtained measures absorbance by microplate reader.With the concentration of Ractopamine in colorimetric analysis result determination testing sample.
Specific embodiment three
The enzyme-linked immunologic detecting kit applying above-mentioned detection Ractopamine to detect a method for Ractopamine concentration, with Fe 3o 4@β-CD magnetic nanoparticle catches the RAC in solution as capture probe, adopt the antibody that the method for noncompetitive immunity makes the RAC specific bond on capture probe add, by washing plate and magnetic resolution removes free antigen-antibody bond and other educt; Add colloidal platinum mark PV and antibody specific bond again, through hatching and washing version, leave the immune complex of Ractopamine antigen, antibody, colloidal platinum mark PV composition.The amount of this immune complex reduces with the increase of Ractopamine in sample.Finally add DAB colour developing, carry out colorimetric analysis.With the concentration (principle as shown in Figure 6) of Ractopamine in colorimetric analysis result determination testing sample.
PtNPs colloidal solid itself has obvious catalyzed coloration effect to chromogenic substrate DAB; PV enzyme reagent gathers multienzyme complex as a kind of height of the HRP of being rich in enzyme simultaneously, and HRP a large amount of on it can effectively develop the color by catalysis DAB, and compared to traditional enzyme labelled antibody, PV enzyme reagent has extremely strong enlarge-effect; In addition, PtNPs colloidal solid has very strong biocompatibility, can combine with PV but not affect the catalytic effect of PV.
Specific embodiment four
The sensitivity that the kit for testing lecdopamine ELISA that the present invention is based on PtNPs/PV detects Ractopamine and the range of linearity
Using utilize specific embodiment two method to prepare PtNPs/PV compound as signal probe, and add the standard items of variable concentrations according to immune complex preparation process and wash plate, standard solution concentration is followed successively by: 0.001 ng mL -1, 0.05 ng mL -1, 0.1 ng mL -1, 0.3 ng mL -1, 0.9 ng mL -1, 2.7 ng mL -1, 8.1 ng mL -1, 20 ng mL -1, 40 ng mL -1, 60 ng mL -1, 80 ng mL -1(standard items solvent is PBS buffer solution), adds A liquid and each 50 μ L of B liquid of DAB in every hole of the kit for testing lecdopamine ELISA obtained, lucifuge is hatched the 15-30min(concrete time and is as the criterion with chromogenic reaction intensity); The DAB color product obtained measures absorbance by microplate reader, and obtain the typical curve of competitive experiment, curvilinear equation is y=0.17884 log C rAC+ 1.08753; R 2=0.98355 as shown in Figure 7.This immune colourimetry is 0.001-50 ng mL to the sensing range of Ractopamine -1; This experiment favorable reproducibility, stable performance, makes simply and easily upgrades.
From measuring, the generally commercially available detection of competitive immunization colourimetry to Ractopamine is limited to 0.1 ng mL -1, what this programme proposed is limited to 0.001 ng mL with PtNPs/PV compound as the detection of competitive immunization colourimetry to Ractopamine of signal probe -1, improve about 10 times than the detectability of commercially available kit, absolutely proved that this scheme has larger advantage and larger using value than commercially available Ractopamine detection kit.
Specific embodiment five
High selectivity confirmatory experiment
In order to assess the specificity of the method for proposed colloidal platinum-polymerase dual amplification colorimetric determination Ractopamine, we will comprise salbutamol (SAL), and the multiple β-agonists such as Clenbuterol (CLE) and dopamine (DOA) detect as interfering material.As can be known from the results, the salbutamol (SAL) that homogenous quantities is simple in optimal conditions, Clenbuterol (CLE) and dopamine (DOA) have no significant effect this immune response result, and the mixing of these chaff interferences also has no significant effect testing result, cross reaction result is all less than 1%.Result shows that the Real-Time Monitoring of proposed immunization method to Ractopamine has high specificity.
Specific embodiment six
Precise 1.0 ± 0.05g pork sample, loads centrifuge tube, adds the phosphate buffer solution of 50mL 0.01 mol/L pH=7.4 in centrifuge tube, uses the abundant whirling motion mixing of high speed disperser; Add appropriate Ractopamine respectively, sonic oscillation 15min, then centrifugal 5min at 4 DEG C, gets supernatant, and adding 2M sodium hydroxide solution adjust ph is 6.5-7.5, leaves standstill 5min, gets supernatant 20 μ L for analyzing.Add A liquid and each 50 μ L of B liquid of DAB in every hole of the kit for testing lecdopamine ELISA prepared by above-mentioned specific embodiment two, lucifuge is hatched the 15-30min(concrete time and is as the criterion with chromogenic reaction intensity); The DAB color product obtained measures absorbance by microplate reader, and result is as shown in table 1.
The testing result of Ractopamine in table 1 pork
From table 1 testing result, the average recovery rate of the method for the colloidal platinum-polymerase dual amplification colorimetric determination Ractopamine proposed is 94.0 ~ 106%, show that the kit for testing lecdopamine ELISA prepared based on the present invention is high for the detection precision of Ractopamine, result accurately and reliably.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.

Claims (6)

1. for detecting an enzyme-linked immunologic detecting kit for Ractopamine, comprising ELISA Plate and signal probe, it is characterized in that: described ELISA Plate is fixed with Ractopamine antigen and capture probe Fe 3o 4@β-CD, described signal probe is the compound of the PtNPs/PV of a large amount of PtNPs of load, and standard items are Ractopamine standard items, and antibody working fluid is anti-Anti-ractopamine antibody monoclonal antibody, nitrite ion A, nitrite ion B are developer DAB, and stop buffer is 2M HCl or 2M H 2sO 4, washing lotion is PBST damping fluid.
2., for detecting a preparation method for the enzyme-linked immunologic detecting kit of Ractopamine, it is characterized in that concrete steps are as follows:
(1) Fe 3o 4-NH 2preparation
By anhydrous sodium acetate, hexane diamine and FeCl 36 H 2join in ethylene glycol after O mixing, stir under 42-60 DEG C of condition and obtain source of iron solution, source of iron solution is transferred in muffle furnace, react 6-8h under 195-205 DEG C of condition after, be separated with magnet, use water and alcohol flushing 2-3 time more respectively, remove the solvent of non-complete reaction, obtain the Fe that particle diameter is 15-25 nm 3o 4-NH 2magnetic nanoparticle;
(2) Fe 3o 4the preparation of@β-CD
Adding to containing carboxymethylation beta-schardinger dextrin-mass concentration by N-Hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine is in the PBS solution of pH 7.4 of 1wt%, at room temperature stir after spending the night, then add the Fe that step (1) prepares 3o 4-NH 2magnetic nanoparticle, after stirring 5-7h continuously, is separated with magnet, more repeatedly cleans with deionized water, to remove unreacted chemical substance, and the Fe obtained 3o 4@β-CD;
(3) preparation of PtNPs
The platinum acid chloride solution of 2ml 1wt% is joined after gently stirring 4-6min in beaker, add rapidly the sodium borohydride solution reduction of 5ml 10mmol/L, vigorous stirring under room temperature, by removing impurity in the centrifugal 15-25min of 10000-12000r/min after reaction terminates, be settled to 2ml after washing at least 3 times and obtain PtNPs solution, save backup under 4 DEG C of conditions; Wherein beaker soaks through chloroazotic acid;
(4) preparation of PtNPs/PV complex signal label
PtNPs solution gentle agitation 5-10min at 0-4 DEG C that step (3) is prepared, slowly drip equal-volume PV enzyme reagent under agitation, stir the bovine serum albumen solution adding 1wt% after spending the night, 2-6 hour is kept in low temperature 0-4 DEG C, namely obtain PtNPs/PV complex solution, save backup under 4 DEG C of conditions;
(5) preparation of enzyme linked immunological kit immune complex
5 mgFe are added in every hole of ELISA Plate 3o 4the standard items of the Ractopamine of@β-CD and 50 μ L serial dilutions, the anti-Anti-ractopamine antibody solution of 50 μ L, mix rear 37 DEG C of lucifuges and hatch 30min, PBST damping fluid washes plate 3 times, and magnet is separated, and then every hole adds 80 μ L PtNPs/PV complex solutions, mix rear 37 DEG C of lucifuges and hatch 30min, PBST damping fluid washes plate 3 times, and magnet is separated, and namely obtains kit for testing lecdopamine ELISA; Wherein the dilution of Ractopamine standard items is 0.01 mol L -1the PBS solution of pH 7.2-7.4, wherein PBST damping fluid is the 0.01 mol L containing 0.05 v/v % tween -1the PBS solution of pH 7.2-7.4.
3. the preparation method of a kind of enzyme-linked immunologic detecting kit for detecting Ractopamine according to claim 2, is characterized in that: anhydrous sodium acetate, hexane diamine, FeCl in step (1) 36 H 2the blending ratio of O and ethylene glycol is 4g:13g:2g:30ml.
4. the preparation method of a kind of enzyme-linked immunologic detecting kit for detecting Ractopamine according to claim 2, is characterized in that: N-Hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, carboxymethylation beta-schardinger dextrin-and Fe in step (2) 3o 4-NH 2the mass ratio of magnetic nanoparticle is 1:1:2:1.
5. the preparation method of a kind of enzyme-linked immunologic detecting kit for detecting Ractopamine according to claim 2, it is characterized in that: the A liquid and each 50 μ L of B liquid that add diaminobenzidine in every hole of the kit for testing lecdopamine ELISA obtained in step (5), lucifuge hatches 15-30min, and the DAB color product obtained measures absorbance by microplate reader.
6. application rights requires that the enzyme-linked immunologic detecting kit of the detection Ractopamine according to any one of 1-5 detects a method for Ractopamine concentration, it is characterized in that concrete steps are as follows: with Fe 3o 4@β-CD magnetic nanoparticle catches the Ractopamine in solution as capture probe, adopt the antibody that the method for noncompetitive immunity makes the RAC specific bond on capture probe add, by wash plate and magnetic resolution remove free antigen-antibody bond and other educt only more than be fixed on antibody at the bottom of plate; Add colloidal platinum mark PV and antibody specific bond again, through hatching and washing plate, leave the immune complex of Ractopamine antigen, antibody, colloidal platinum mark PV composition, the amount of this immune complex increases with the increase of Ractopamine in sample, finally add DAB colour developing, carry out colorimetric analysis, with the concentration of Ractopamine in colorimetric analysis result determination testing sample.
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