CN104655848B - 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 PDFInfo
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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
Technical field
The present invention relates to Ractopamine detection technique, especially relate to one kind and be based on fe3o4@β-cd as capture probe,
Ptnps/pv is as signal probe for detecting enzyme-linked immunologic detecting kit of Ractopamine and preparation method thereof and answering
With.
Background technology
β2Receptor stimulating agent (β-adrenergic agonists) because symptoms of asthma can be alleviated, thus being often used as veterinary drug；
Muscle, minimizing lipopexia etc. can be increased also often be added in feedstuff yet with it.Ractopamine (ractopamine,
Rac) as β2A member of receptor stimulating agent is a kind of Ke Lunbaan of synthetic, just by as a kind of new clenbuterol hydrochloride one
A little pig farms use.The Ministry of Agriculture of China, Ministry of Public Health, state food drug surveilance office combine " forbidding in feedstuff and animal of issue
Types of drugs catalogue used in drinking water " in provide against Ractopamine and use in feedstuff as growth promoter.Cause
This, need a kind of quick, high-sensitive Ractopamine trace residue detection method badly as the important means ensureing food safety.
At present, substantial amounts of detection meanss have been studied and have been used for the detection of Ractopamine, wherein more such as
Gas chromatogram (gc), liquid chromatograph (lc) and they with mass spectrometry (gc-ms, lc-ms).But all there is instrumentation in them
Complexity, expensive equipment, the problems such as time-consuming, these problems make them be difficult to on-site quick screening.Quick detection test paper
Though bar convenient be only capable of accomplishing qualitative it is impossible to quantitative.Meanwhile, traditional Ractopamine elisa detection kit typically makes
With competition law although effective and rapid, but still suffer from that test limit is too high, false positive the shortcomings of.Also do not disclose both at home and abroad at present and appoint
What is with regard to utilizing fe3o4@β-cd is used for detecting Ractopamine as enzyme labelled antibody as capture probe, ptnps/pv complex
Immune colorimetry correlational study report.
Content of the invention
The technical problem to be solved be provide that a kind of detection speed is fast, accuracy and sensitivity is high, specificity
Strong and easily operated for detecting enzyme-linked immunologic detecting kit of Ractopamine and its preparation method and application.
The technical scheme that present invention solution above-mentioned technical problem is adopted is: a kind of enzyme for detecting Ractopamine joins
Immunity detection reagent, including ELISA Plate and signal probe, described ELISA Plate is contained within Ractopamine antigen and capture is visited
Pin fe3o4@β-cd, described signal probe is the complex of the ptnps/pv having loaded a large amount of ptnps, and standard substance are that Rec is many
Bar amine standard substance, antibody working solution is anti-Anti-ractopamine antibody monoclonal antibody, and nitrite ion is a liquid in dab color development system
With b liquid, terminate liquid is 2m hcl or 2m h2so4, washing liquid is pbst buffer.
A kind of preparation method of the enzyme-linked immunologic detecting kit for detecting Ractopamine, specifically comprises the following steps that
(1)fe3o4-nh2Preparation
By anhydrous sodium acetate, hexamethylene diamine (1,6-hexanediamine) and fecl3·6h2It is added to ethylene glycol after o mixing
In, stirring under the conditions of 42-60 DEG C obtains source of iron solution, source of iron solution is transferred in Muffle furnace, under the conditions of 195-205 DEG C
After reaction 6-8h, separated with Magnet, then use water and alcohol flushing 2-3 time respectively, the solvent of removing not reaction completely, obtain particle diameter
Fe for 15-25nm3o4-nh2Magnetic nanoparticle；
(2)fe3o4The preparation of@β-cd
N-Hydroxysuccinimide (nhs) and 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine (edc) are added to
Beta-schardinger dextrin-containing carboxy methylation (carboxymethyl- β-cd, cm- β-cd) mass concentration is the pbs solution (ph=of 1wt%
7.4), in, after being stirred at room temperature overnight (to activate the carboxymethyl on beta-schardinger dextrin -), add what step (1) prepared
fe3o4-nh2Magnetic nanoparticle, after continuous stirring 5-7h, is separated with Magnet, then deionized water is cleaned repeatedly, to remove not
The chemical substance of reaction, the fe obtaining3o4@β-cd；
(3) preparation of ptnps
The platinum acid chloride solution of 2ml 1wt% is added in beaker and gently stirs after 4-6min, be rapidly added 5ml 10mmol/l
Sodium borohydride solution reduction, be stirred vigorously under room temperature, reaction terminate after by 10000-12000r/min be centrifuged 15-
25min removes impurity, is settled to 2ml and obtains ptnps solution, save backup under the conditions of 4 DEG C after washing at least 3 times；Wherein burn
Cup soaks through chloroazotic acid；
(4) preparation of ptnps/pv complex signal label
The ptnps solution that step (3) is prepared is at 0-4 DEG C (in order that temperature is unlikely to when pv enzyme is added
Too high and make enzyme inactivate) under gentle agitation 5-10min, be slowly added dropwise equal-volume pv enzymatic reagent under agitation
(powervision, purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), adds the Ox blood serum egg of 1wt% after being stirred overnight
White solution, in low temperature, 0-4 DEG C keeps 2-6 hour (unnecessary key mapping on closing ptnps), that is, obtain ptnps/pv complex molten
Liquid, saves backup under the conditions of 4 DEG C；
(5) preparation of enzyme linked immunological kit immune complex
5mgfe is added in every hole of ELISA Plate3o4The standard substance of the Ractopamine that@β-cd and 50 μ l is serially diluted
(0.03、0.1、0.3、0.9、2.7、8.1ng ml-1), the anti-Anti-ractopamine antibody solution of 50 μ l, mix rear 37 DEG C of lucifuges incubation
30min, pbst buffer (the 0.01mol l containing 0.05v/v% tween-1The pbs solution of ph 7.2-7.4) wash plate 3 times, magnetic
Ferrum separates, and then every hole adds 80 μ l ptnps/pv complex solutions, mixes rear 37 DEG C of lucifuges and is incubated 30min, pbst buffer
Wash plate 3 times, Magnet separates, that is, obtain Ractopamine elisa detection kit；The diluent of wherein Ractopamine standard substance
For 0.01mol l-1Ph 7.2-7.4 pbs solution (pbs phosphate buffer be purchased from Beijing Suo Laibao Science and Technology Ltd:
0.01m ph 7.2-7.4).
Anhydrous sodium acetate, hexamethylene diamine (1,6-hexanediamine), fecl in step (1)3·6h2O and ethylene glycol mixed
Composition and division in a proportion example is 4g:13g:2g:30ml.
N-Hydroxysuccinimide, 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine, carboxy methylation in step (2)
Beta-schardinger dextrin-and fe3o4-nh2The mass ratio of magnetic nanoparticle is 1:1:2:1.
Diaminobenzidine is added in every hole of the Ractopamine elisa detection kit obtaining in step (5)
(dab nitrite ion is the medicine inside Ractopamine detection kit, and Ractopamine test kit is purchased from Beijing Huaan Mai Kesheng
Thing Technology Co., Ltd., production code member: he09003) a liquid and each 50 μ l of b liquid, lucifuge is incubated 15-30min, obtained dab
Color product passes through microplate reader mensuration absorbance.
A kind of enzyme-linked immunologic detecting kit applying above-mentioned detection Ractopamine detects the side of Ractopamine concentration
Method, specifically comprises the following steps that with fe3o4@β-cd magnetic nanoparticle captures the Ractopamine in solution as capture probe
(rac), the antibody that the rac specific bond on capture probe added is made using the method for noncompetitive immunity, by wash plate and
Magnetic Isolation removes free antibody and other only remaining antibody being fixed on plate bottom of educt；Add colloidal platinum labelling pv and resist
Body specific bond, through being incubated and washing plate, leaves Ractopamine antigen, antibody, the immune complex of colloidal platinum labelling pv composition,
The amount of this immune complex increases with the increase of Ractopamine in sample, is eventually adding dab colour developing, carries out colorimetric analysiss,
Determine the concentration of Ractopamine in testing sample with colorimetric analysiss result.
Compared with prior art, it is an advantage of the current invention that: present invention firstly discloses detection Ractopamine enzyme connection
Immunity detection reagent and its preparation method and application, powervision (pv) reagent is a kind of polymerase chelate, containing anti-
Body and a large amount of horseradish peroxidase (hrp), compared to traditional enzyme labelled antibody, this reagent increased labelling on unit antibody
Hrp enzyme, reaches the purpose of signal amplification by the method increasing hrp enzyme content.Meanwhile, Pt colloids granule (ptnps) is urged to hrp
Changing substrate diaminobenzidine (diaminobenzidine, dab) has a very strong catalytic effect, and this catalytic effect and ptnps
Addition is in proportionate relationship.And, ptnps synthesis step is simple, particle stabilized, have very strong biocompatibility, can be with enzyme egg
Combine in vain and do not interfere with its catalytic effect, can be used to load protease.
The present invention uses beta-schardinger dextrin-(β-cyclodextrins, β-cd) to capture trace in sample liquid as capture probe
Ractopamine., as a kind of Subjective and Objective material, its inclusion technique is further wide in medicine and Applications in Food Industry for beta-schardinger dextrin-
General, in its energy selective absorption sample liquid phenol structure, Ractopamine two end is symmetric phenol structure, so as to have
Imitate to enter beta-schardinger dextrin-inner chamber and realize antigen and fix.Because beta-schardinger dextrin-is a kind of soluble polysaccharide, divide to realize sample
From purpose, by (fe on grafted by beta cyclodextrin to amination ferroso-ferric oxide3o4-nh2), reached quick, simple using externally-applied magnetic field
Single separation.fe3o4-nh2Particle diameter is less, does not have characteristic absorption peak in the range of ultraviolet 230-1000nm, the mensure to colour developing result
Do not affect.
In sum, the present invention detects enzyme-linked immunologic detecting kit of Ractopamine and its preparation method and application,
Using fe3o4@β-cd as capture probe, ptnps/pv as signal probe immune colorimetric determination Rct opamine residue.
fe3o4@β-cd is as trace rac in a large amount of enrichment solutions of capture probe energy；Ptnps colloidal solid itself has to chromogenic substrate dab
Significantly catalyzed coloration effect；Pv enzymatic reagent is combined as a kind of poly- enzyme of height rich in horseradish peroxidase (hrp enzyme) simultaneously
Thing, thereon substantial amounts of hrp can effectively be catalyzed dab colour developing, 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 passes through
Replacing the enzyme labelled antibody of conventional reagents box, co-catalysis substrate dab produces color change to ptnps labelling pv, dual to reach
Amplification effect, thus realizing reliable, quick, sensitive, the high specific detection of Ractopamine trace residue in food, thus
The aspects such as food, medical science have its huge using value.
Brief description
Fig. 1 is the fe of preparation3o4-nh2Scanning electron microscope (SEM) photograph；
Fig. 2 is the fe of preparation3o4The 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 complex of preparation；
Fig. 5 is the phenogram preparing ptnps/pv complex；The ultraviolet-visible absorption spectroscopy of the ptnps of a: preparation；B: many
The ultraviolet-visible absorption spectroscopy of poly- multienzyme complex pv；The ultraviolet-visible absorption spectroscopy of the ptnps/pv complex of c: preparation；
Fig. 6 is the schematic diagram based on ptnps/pv as signal probe immunity colorimetric determination Ractopamine；
Fig. 7 is the canonical plotting that object Ractopamine light absorption value is set up with Ractopamine concentration.
Specific embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
A kind of enzyme linked immunological kit for detecting Ractopamine, including ELISA Plate and signal probe, in ELISA Plate
Containing Ractopamine antigen and capture probe fe3o4@β-cd, signal probe is the answering of ptnps/pv having loaded a large amount of ptnps
Compound, standard substance are Ractopamine standard substance, and antibody working solution is anti-Anti-ractopamine antibody monoclonal antibody, and nitrite ion is
A liquid in dab color development system and b liquid, terminate liquid is 2m hcl or 2m h2so4, washing liquid is pbst buffer.
This colorimetry is mainly by fe3o4@β-cd magnetic nanoparticle captures the Rec DOPA in solution as capture probe
Amine (rac), and the antibody that the rac specific bond on capture probe added, this antibody are made using the method for noncompetitive immunity
Antibody on energy specific bond ptnps/pv complex, through incubation and Magnetic Isolation, stays capture probe, Ractopamine to resist
The former, immune complex of antibody, ptnps/pv composition.The amount of this immune complex increases with the increase of Ractopamine in sample
Plus, linear with the Ractopamine in sample.It is eventually adding dab colour developing, carry out colorimetric analysiss.With colorimetric analysiss result
Determine the concentration of Ractopamine in testing sample.
Specific embodiment two
A kind of preparation method of the enzyme-linked immunologic detecting kit for detecting Ractopamine, specifically comprises the following steps that
(1)fe3o4-nh2Preparation
By anhydrous sodium acetate, hexamethylene diamine (1,6-hexanediamine) and fecl3·6h2It is added to ethylene glycol after o mixing
In, stirring under the conditions of 42-60 DEG C obtains source of iron solution, source of iron solution is transferred in Muffle furnace, under the conditions of 195-205 DEG C
After reaction 6-8h, separated with Magnet, then use water and alcohol flushing 2-3 time respectively, the solvent of removing not reaction completely, obtain particle diameter
Fe for 15-25nm3o4-nh2Magnetic nanoparticle；Wherein anhydrous sodium acetate, hexamethylene diamine (1,6-hexanediamine),
fecl3·6h2O and the mixed proportion pressing ethylene glycol are 4g:13g:2g:30ml；Fig. 1 is the fe of preparation3o4-nh2Scanning electron microscope
Figure；Prepared magnetic fe3o4-nh2The particle diameter of granule is about 17nm, and it is rough, in a large amount of gullies shape, this structure
Make this magnetic fe3o4-nh2There is the load for β-cd for the great surface area, thus improving the adsorptivity of capture probe；
(2)fe3o4The preparation of@β-cd
N-Hydroxysuccinimide (nhs) and 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine (edc) are added to
Beta-schardinger dextrin-containing carboxy methylation (carboxymethyl- β-cd, cm- β-cd) mass concentration is the pbs solution (ph=of 1wt%
7.4), in, after being stirred at room temperature overnight (to activate the carboxymethyl on beta-schardinger dextrin -), add what step (1) prepared
fe3o4-nh2Magnetic nanoparticle, after continuous stirring 5-7h, is separated with Magnet, then deionized water is cleaned repeatedly, to remove not
The chemical substance of reaction, the fe obtaining3o4@β-cd；Wherein N-Hydroxysuccinimide, 1- ethyl-(3- dimethylaminopropyl) carbon
Acyl diimine, carboxy methylation beta-schardinger dextrin-and fe3o4-nh2The mass ratio of magnetic nanoparticle is 1:1:2:1；Fig. 2 is preparation
fe3o4The scanning electron microscope (SEM) photograph of@β-cd；Contrast Fig. 4 can be seen that after combining β-cd, and the particle diameter of this magnetic-particle increases,
This illustrates capture probe fe3o4@β-cd successfully synthesizes；
(3) preparation of ptnps
The platinum acid chloride solution of 2ml 1wt% is added in beaker and gently stirs after 4-6min, be rapidly added 5ml 10mmol/l
Sodium borohydride solution reduction, be stirred vigorously under room temperature, reaction terminate after 10000-12000r/min centrifugation 15-25min remove
Decontamination, is settled to 2ml after washing at least 3 times and obtains ptnps solution, save backup under the conditions of 4 DEG C；Wherein beaker is through chloroazotic acid
Soak；Wherein beaker soaks through chloroazotic acid；Fig. 3 is the transmission electron microscope picture of the ptnps of preparation, and prepared ptnps granule presents relatively
Uniformly spherical, its particle diameter is about 8nm；
(4) preparation of ptnps/pv complex signal label
The ptnps solution that step (3) is prepared is at 0-4 DEG C (in order that temperature is unlikely to when pv enzyme is added
Too high and make enzyme inactivate) under gentle agitation 5-10min, be slowly added dropwise under agitation equal-volume pv enzymatic reagent be purchased from Beijing in
Bioisystech Co., Ltd of China fir Golden Bridge, model: pv-6000, specification: 3ml, English name: polymer detection system
For immuno-histological staining, is powervision in document), add the cattle of 1wt% after being stirred overnight
Serum albumin solution, in low temperature, 0-4 DEG C keeps 2-6 hour (unnecessary key mapping on closing ptnps), that is, obtain ptnps/pv and be combined
Thing solution, saves backup under the conditions of 4 DEG C；The mixed volume of wherein ptnps solution and powervision reagent is than for 1:1；Figure
4 is the transmission electron microscope picture of the ptnps/pv complex of preparation, and the ptnps granule of contrast Fig. 3 can be seen that ptnps/pv complex
On protein, this explanation ptnps is successfully attached on pv enzyme-antibody multimer chelate middle ptnps even particulate dispersion；Fig. 5
For preparing the phenogram of ptnps/pv complex；The ultraviolet-visible absorption spectroscopy of the ptnps of a: preparation；B: poly multienzyme complex pv
Ultraviolet-visible absorption spectroscopy；The ultraviolet-visible absorption spectroscopy of the ptnps/pv complex of c: preparation；Simple colloid platinum grain
No obvious absorption peaks in the range of 230-1000nm, only have certain absorbance in below 300nm, see Fig. 5 a.Simple pv exists
There is absworption peak, as shown in Figure 5 c at 280nm, 376nm, 506nm.Fig. 5 b is the signal probe after being closed with bsa, and it has simultaneously
Simple colloid platinum grain and the characteristic absorption peak of powervision reagent, its larger absorbing proteins peak is derived partly from
bsa.This explanation colloidal platinum and the ptnps/pv complex that is combined into of powervision reagent successfully synthesize；
(5) preparation of enzyme linked immunological kit immune complex
5mgfe is added in every hole of ELISA Plate3o4The standard substance of the Ractopamine that@β-cd and 50 μ l is serially diluted
(0.03、0.1、0.3、0.9、2.7、8.1ng ml-1), the anti-Anti-ractopamine antibody solution of 50 μ l, mix rear 37 DEG C of lucifuges incubation
30min, pbst buffer (the 0.01mol l containing 0.05v/v% tween-1The pbs solution of ph 7.2-7.4) wash plate 3 times, magnetic
Ferrum separates, and then every hole adds 80 μ l ptnps/pv complex solutions, mixes rear 37 DEG C of lucifuges and is incubated 30min, pbst buffer
Wash plate 3 times, Magnet separates, that is, obtain Ractopamine elisa detection kit；The diluent of wherein Ractopamine standard substance
For 0.01mol l-1The pbs solution (pbs phosphate buffer is purchased from Beijing Suo Laibao Science and Technology Ltd) of ph 7.2-7.4.
(dab nitrite ion is that Rec is many to add diaminobenzidine in every hole of Ractopamine elisa detection kit
Medicine inside bar amine detection kit, Ractopamine test kit is purchased from Beijing Huaan Magnech Bio-Tech Co., Ltd., produces
Product are numbered: a liquid he09003) and each 50 μ l of b liquid, and lucifuge is incubated 15-30min, and obtained dab color product passes through enzyme mark
Instrument mensuration absorbance.Determine the concentration of Ractopamine in testing sample with colorimetric analysiss result.
Specific embodiment three
A kind of enzyme-linked immunologic detecting kit applying above-mentioned detection Ractopamine is detecting Ractopamine concentration
Method, with fe3o4@β-cd magnetic nanoparticle captures the rac in solution as capture probe, using the side of noncompetitive immunity
Method makes the antibody that the rac specific bond on capture probe is added, by wash plate and Magnetic Isolation remove free antibody and its
Its educt；Add colloidal platinum labelling pv and antibody specific bond, through being incubated and washing version, leave Ractopamine antigen, resist
Body, the immune complex of colloidal platinum labelling pv composition.The amount of this immune complex increases with the increase of Ractopamine in sample
Plus.It is eventually adding dab colour developing, carry out colorimetric analysiss.Determine the concentration of Ractopamine in testing sample with colorimetric analysiss result
(principle is as shown in Figure 6).
Ptnps colloidal solid itself has obvious catalyzed coloration effect to chromogenic substrate dab；Pv enzymatic reagent is as one simultaneously
Plant the poly- multienzyme complex of height rich in hrp enzyme, substantial amounts of hrp can effectively be catalyzed dab colour developing thereon, resists compared to traditional enzyme mark
Body, pv enzymatic reagent has extremely strong enlarge-effect；In addition, ptnps colloidal solid has very strong biocompatibility, can with pv combine but not
The catalytic effect of impact pv.
Specific embodiment four
The sensitivity that the present invention is detected to Ractopamine based on the Ractopamine elisa detection kit of ptnps/pv
And the range of linearity
Using the ptnps/pv complex prepared by the use of specific embodiment two method as signal probe and compound according to immunity
Thing preparation process adds the standard substance of variable concentrations and washes plate, and standard solution concentration is followed successively by: 0.001ng ml-1、0.05ng
ml-1、0.1ng ml-1、0.3ng ml-1、0.9ng ml-1、2.7ng ml-1、8.1ng ml-1、20ng ml-1、40ng ml-1、
60ng ml-1、80ng ml-1(standard substance solvent is pbs buffer solution), the Ractopamine elisa detection kit obtaining
A liquid and each 50 μ l of b liquid of dab, lucifuge incubation 15-30min (the concrete time is defined) is added by chromogenic reaction intensity in every hole；Institute
The dab color product obtaining passes through microplate reader mensuration absorbance, obtains the standard curve of non-competitive assay, and curvilinear equation is y
=0.17884log crac+1.08753；r2=0.98355 is as shown in Figure 7.This immune colorimetry detection to Ractopamine
Scope is 0.001-50ng ml-1；This experiment favorable reproducibility, stable performance, make and simply and readily update.
From measuring, typically commercially available competitive immunization colorimetry is limited to 0.1ng to the detection of Ractopamine
ml-1, this programme propose use ptnps/pv complex as signal probe noncompetitive immunity colorimetry to Ractopamine
Detection be limited to 0.001ng ml-1, improve 10 times about than the test limit of commercially available test kit, absolutely proved this scheme ratio
Commercially available Ractopamine detection kit has bigger advantage and bigger using value.
Specific embodiment five
High selectivity confirmatory experiment
In order to assess proposed colloidal platinum-polymerase dual amplification colorimetric determination Ractopamine method special
Property, we will include albuterol (sal), and multiple β-agonists such as Clenbuterol (cle) and dopamine (doa) is as interference
Material is detected.As can be known from the results, the simple albuterol of homogenous quantities (sal) in optimal conditions, Clenbuterol (cle) and
Dopamine (doa) has no significant effect to this immunoreation result, and the mixing of these chaff interferences does not have to testing result yet
Appreciable impact, cross reaction result is respectively less than 1%.Result shows the real-time monitoring to Ractopamine for the proposed immunization method
There is high specificity.
Specific embodiment six
Precise 1.0 ± 0.05g Carnis Sus domestica sample, loads centrifuge tube, adds 50ml 0.01mol/l ph in centrifuge tube
=7.4 phosphate buffer solution, is mixed using the abundant whirling motion of high speed disperser；It is separately added into appropriate Ractopamine, ultrasonic
Vibration 15min, is then centrifuged 5min at 4 DEG C, takes supernatant, and adding 2m sodium hydroxide solution to adjust ph value is 6.5-7.5, quiet
Put 5min, take supernatant 20 μ l to be used for analyzing.The Ractopamine elisa detection examination that above-mentioned specific embodiment two is prepared
The a liquid of dab and each 50 μ l of b liquid are added, (the concrete time with chromogenic reaction intensity is lucifuge incubation 15-30min in every hole of agent box
Accurate)；Obtained dab color product passes through microplate reader mensuration absorbance, and result is as shown in table 1.
The testing result of Ractopamine in table 1 Carnis Sus domestica
From table 1 testing result, the colloidal platinum being proposed-polymerase dual amplification colorimetric determination Ractopamine
The average recovery rate of method is 94.0～106%, shows the Ractopamine elisa detection kit pair based on present invention preparation
High in the detection precision of Ractopamine, result is accurately and reliably.
Certainly, described above not limitation of the present invention, the present invention is also not limited to the example above.The art
Those of ordinary skill, in the essential scope of the present invention, the change made, remodeling, adds or replaces, and also should belong to the present invention's
Protection domain.
Claims (2)
1. a kind of preparation method of the enzyme-linked immunologic detecting kit for detecting Ractopamine is it is characterised in that concrete steps
As follows:
(1)fe3o4-nh2Preparation
By anhydrous sodium acetate, hexamethylene diamine and fecl3·6h2It is added in ethylene glycol after o mixing, stir under the conditions of 42-60 DEG C
To source of iron solution, source of iron solution is transferred in Muffle furnace, after reaction 6-8h under the conditions of 195-205 DEG C, separated with Magnet, then
Use water and alcohol flushing 2-3 time respectively, remove the solvent of not reaction completely, obtain the fe for 15-25nm for the particle diameter3o4-nh2Magnetic
Nano-particle；Wherein anhydrous sodium acetate, hexamethylene diamine, fecl3·6h2The mixed proportion of o and ethylene glycol is 4g:13g:2g:30ml；
(2)fe3o4The preparation of@β-cd
N-Hydroxysuccinimide and 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine are added to β containing carboxy methylation-ring
Dextrin mass concentration is in the pbs solution of ph 7.4 of 1wt%, after being stirred at room temperature overnight, adds step (1) and is prepared into
The fe arriving3o4-nh2Magnetic nanoparticle, after continuous stirring 5-7h, is separated with Magnet, then deionized water is cleaned, repeatedly to remove
Go unreacted chemical substance, the fe obtaining3o4@β-cd；Wherein N-Hydroxysuccinimide, 1- ethyl-(3- dimethylamino third
Base) phosphinylidyne diimine, carboxy methylation beta-schardinger dextrin-and fe3o4-nh2The mass ratio of magnetic nanoparticle is 1:1:2:1；
(3) preparation of ptnps
The platinum acid chloride solution of 2ml 1wt% is added in beaker and gently stirs after 4-6min, be rapidly added the boron of 5ml 10mmol/l
Sodium hydride solution reduces, and is stirred vigorously under room temperature, and reaction is removed by being centrifuged 15-25min in 10000-12000r/min after terminating
Decontamination, is settled to 2ml after washing at least 3 times and obtains ptnps solution, save backup under the conditions of 4 DEG C；Wherein beaker is through chloroazotic acid
Soak；
(4) preparation of ptnps/pv complex signal label
The ptnps solution that step (3) is prepared gentle agitation 5-10min at 0-4 DEG C, is slowly added dropwise under agitation
Equal-volume pv enzymatic reagent, adds the bovine serum albumen solution of 1wt% after being stirred overnight, in low temperature, 0-4 DEG C keeps 2-6 hour, that is,
Obtain ptnps/pv complex solution, save backup under the conditions of 4 DEG C；
(5) preparation of enzyme linked immunological kit immune complex
5mgfe is added in every hole of ELISA Plate3o4The standard substance of the Ractopamine that@β-cd and 50 μ l is serially diluted, 50 μ l resist
Anti-ractopamine antibody solution, mixes rear 37 DEG C of lucifuges and is incubated 30min, and plate 3 times washed by pbst buffer, and Magnet separates, then often
Hole adds 80 μ l ptnps/pv complex solutions, mixes rear 37 DEG C of lucifuges and is incubated 30min, and pbst buffer washes plate 3 times, Magnet
Separate, that is, obtain Ractopamine elisa detection kit；The diluent of wherein Ractopamine standard substance is 0.01mol l- 1The pbs solution of ph 7.2-7.4, wherein pbst buffer are the 0.01mol l containing 0.05v/v% tween-1ph 7.2-7.4
Pbs solution.
2. the preparation side of a kind of enzyme-linked immunologic detecting kit for detecting Ractopamine according to claim 1
Method it is characterised in that: in every hole of the Ractopamine elisa detection kit obtaining in step (5) add diaminourea connection
The a liquid of aniline and each 50 μ l of b liquid, lucifuge is incubated 15-30min, and obtained dab color product measures extinction by microplate reader
Degree.
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| CN109682871B (en) * | 2019-01-16 | 2021-04-16 | 扬州大学 | beta-CD @ CdS nanorod applied to photoelectric biosensing and preparation method thereof |
| CN112206824A (en) * | 2020-10-30 | 2021-01-12 | 江西维邦生物科技有限公司 | Preparation method of polydopamine-mediated magnetic bimetallic nanoenzyme |
| CN113933500A (en) * | 2021-09-30 | 2022-01-14 | 山东省农业科学院原子能农业应用研究所(山东省辐照中心、山东省农业科学院农产品研究所) | Portable high-sensitivity immunoassay kit for ractopamine and application thereof |
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| CN102350312A (en) * | 2011-07-19 | 2012-02-15 | 山西大学 | Beta-cyclodextrin functionalized Fe3O4 magnetic nanometer adsorbent and use thereof |
| CN103041782A (en) * | 2012-11-22 | 2013-04-17 | 中国农业科学院农业质量标准与检测技术研究所 | Functionalized Fe3O4 nano-particle and preparation method and application thereof |
| CN103406081A (en) * | 2013-07-16 | 2013-11-27 | 商洛学院 | Preparation method and applications of anion beta-cyclodextrin magnetic microsphere |
| CN103792328A (en) * | 2014-01-13 | 2014-05-14 | 宁波大学 | Method for detecting ractopamine content in food |
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| CN103041782A (en) * | 2012-11-22 | 2013-04-17 | 中国农业科学院农业质量标准与检测技术研究所 | Functionalized Fe3O4 nano-particle and preparation method and application thereof |
| CN103406081A (en) * | 2013-07-16 | 2013-11-27 | 商洛学院 | Preparation method and applications of anion beta-cyclodextrin magnetic microsphere |
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Application publication date: 20150527 Assignee: Ningbo snow Verne Biotechnology Co. Ltd. Assignor: Ningbo University Contract record no.: 2017330000135 Denomination of invention: Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit Granted publication date: 20170118 License type: Common License Record date: 20171018 |