CN107478824A - The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Ractopamine - Google Patents
The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Ractopamine Download PDFInfo
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- CN107478824A CN107478824A CN201710685240.3A CN201710685240A CN107478824A CN 107478824 A CN107478824 A CN 107478824A CN 201710685240 A CN201710685240 A CN 201710685240A CN 107478824 A CN107478824 A CN 107478824A
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/9406—Neurotransmitters
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- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
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Abstract
The invention discloses the magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Ractopamine.The kit includes：Acridinium ester label, coupling have magnetic particle, Ractopamine calibration object, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and the cleaning fluid of antigen or antibody.Kit of the present invention is using Magneto separate chemiluminescence as detection means, in combination with acridinium ester label technology.Magnetism particulate immuno chemistry luminescence method high sensitivity that the present invention establishes, high specificity, accurate quick, detection time is short, and testing result has higher accuracy and repeatability, and the kit is applicable to various luminometer devices.
Description
Technical field
The invention belongs to field of detection of food safety, the immune magnetic microparticle chemiluminescence inspection of specifically a kind of Ractopamine
Test agent box and preparation method.
Background technology
Ractopamine（Ractopamine, RAC）, Chinese nickname is 4- [3- [2- hydroxyls -2- (4- hydroxy phenyls)-second
Base] aminobutyl] phenol, molecular formula C18H23NO3, its structure is：
It is a kind of artificial synthesized Ke Lunbaan beta receptor activators, is beta-stimulants class compound, it is thin to belong to the second generation
Meat essence.Beta-stimulants are called beta-2-agonists, are a kind of chemical constitution adrenaline similar with physiological function and norepinephrine
Phenyl ethylamine class medicine.The 1980s there are some researches show, beta-stimulants influence nutriment flow direction in animal body and
Redistribute, strengthen internal catabolism of fat, protein synthesis increase, significantly improve lean meat percentage.Beta-stimulants remain
It can be gathered in animal edible tissues, because this kind of compound has Orally active, if illegal Use out of range will be to people
The internal organs such as liver, kidney with animal produce toxic side effect.There is research to point out, Excess free enthalpy Ractopamine, human body occurs
Different degrees of toxic reaction, its symptom is similar to animal poisoning symptom, show as muscular tremor, quadriplegia, tachycardia,
The symptoms such as arrhythmia cordis, stomachache, myalgia, nauseous dizziness, severe one can trigger hypertension, heart disease even dead.Therefore wrap
Include European Union, China, Malaysia including more than 150 individual countries prohibit the use of Ractopamine promote growth of animal.
2002,176 bulletins of the Ministry of Agriculture of China, the Ministry of Public Health and National Drug Administration's joint issue《Forbid
The types of drugs catalogue used in feed and animal drinking water》Middle clear stipulaties, forbid Ractopamine to make in animal-breeding
With.No. 110 Department of Commerce, Announcement of the General Administration of Customs 2009 year announcement, from 9 days December in 2009, Rec DOPA of embargoing
Amine and ractopamine hydrochloric.On December 5th, 2011, Ministry of Industry and Information, the Ministry of Agriculture, Department of Commerce, the former Ministry of Public Health, national industrial and commercial administration pipe
Manage general bureau, ministries and commissions of State Administration for Quality Supervision and Inspection and Quarantine six issue joint bulletin 2011 No. 41, it is desirable to from this very day at me
Forbid producing and selling Ractopamine in border.Therefore, Rct opamine residue detects in food, to ensureing that food security rises
Very important effect.
Up to the present, the method for detecting Rct opamine residue in animal derived food mainly has：Efficient liquid phase
Chromatography（HPLC）, gas chromatography-mass spectrography（GC-MS）, liquid chromatograph mass spectrography（LC-MS）, radioimmunology, enzyme
Linked immunosorbent assay（ELISA）, enzyme-catalyzed chemical luminescence etc..But high performance liquid chromatography, gas chromatography-mass spectrography, liquid phase color
Spectrum-MS Instrumental equipment is expensive, and sample pre-treatments are complicated, waste time and energy and are not easy to popularize, and testing cost is high.Radiation
Immunization also needs to be equipped with radioactive source in addition to drawbacks described above, there is certain risk.Although ELISA detection is cheap, fast
Speed, but sensitivity is inadequate, is only applicable to the detection and identification of micro substance, CN 101315379（In December, 2008）Disclose one
Kind is used for kit and its application for detecting Ractopamine, and the kit uses Ractopamine coated elisa plate, horseradish mistake
Oxide enzyme marks Anti-ractopamine antibody, is that sensitivity is low the shortcomings that this method.CN 105759042 A（In July, 2016）It is public
A kind of chemical luminescence ELISA detection kit for detecting Ractopamine is opened, the kit uses Ractopamine Dan Ke
Grand antibody is coated with polystyrene Chemiluminescent plate, and horseradish peroxidase-labeled Ractopamine is made with bovine serum albumin coupling
Artificial antigen, use horseradish peroxidase major defect for：Luminol no horseradish peroxidase presence in the case of,
Also can be by H2O2It is luminous to aoxidize itself, background is of a relatively high, influences signal to noise ratio, kinetics is complicated, and influence factor is more, as a result
It is not sufficiently stable, the substrate that obtain high sensitivity and plateau length is not easy.CN 106568943 A（In April, 2017）Disclose
The detection kit of Ractopamine in a kind of food, the kit is using amino magnetic bead coating Ractopamine, alkaline phosphatase
Enzyme marks Ractopamine associated proteins, use the major defect of alkaline phosphatase for：Substrate reaches the time length of plateau, bottom
Thing cost is high, causes testing cost height.Therefore, a kind of detection Rec effective, quick, simple, sensitive, anti-interference is high is established
The method tool of dopamine is of great significance.
The present invention uses method as direct chemoluminescence method, using acridinium ester as chemiluminescent labels with obvious excellent
Gesture, it is mainly manifested in：Reaction does not need catalyst, as long as alkaline environment can be carried out, is swift in response, can complete catching reaction
Caused photon, background luminescence is low, and signal to noise ratio is high, and disturbing factor is few, and reagent stability is good, and system is simple, and exciting liquid cost is low,
Easily and protein bind, and photon yield is not reduced acridinium ester after being coupled.The Magnetism particulate immuno chemistry luminescence method that the present invention establishes is sensitive
Spend height, high specificity, accurate quick, detection time is short, testing result have higher accuracy with it is repeated.
The content of the invention
It is an object of the invention to provide the Lay that a kind of sensitivity is higher, the reaction time is short, simple to operate, anti-interference is high
The magnetic microparticle chemiluminescence detection kit and preparation method of gram dopamine.
To achieve the above object, the present invention provides following technical scheme：
The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Ractopamine, magnetic particle provided by the present invention
The kit of luminescent method detection Ractopamine is learned, ractopamine monoclonal antibody can be taken to be coupled magnetic particle, acridine
Ester marks Ractopamine antigen, and Ractopamine antigen can also be taken to be coupled magnetic particle, acridinium ester label Ractopamine
Monoclonal antibody.Kit also includes Ractopamine calibration object, the chemiluminescence preexciting liquid A that above-mentioned acridinium ester acts on, changed
Learn luminous exciting liquid B and cleaning fluid.
Described magnetic particle directly can be coupled with antibody or antigen, can be also coupled magnetic particle and Streptavidin, simultaneously
Using biotin labelled antibodies or antigen.
The surface modification group of the magnetic particle is carboxyl, amino, p-toluenesulfonyl or Streptavidin-biotin.
Described chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
Described acridinium ester label is Ractopamine antigen, or ractopamine monoclonal antibody.
Described calibration object is to be with the Tris-HCl buffer solutions containing 0.5-5.0% BSA and 0.1-0.5% PC300
Matrix, add the configuration of Ractopamine sterling and form, calibration object form is liquid.
Described Ractopamine calibration object solution concentration is respectively：0 μg/L、0.02 μg/L、0.1 μg/L、0.5 μ
g/L、2.5 μg/L、12.5 μg/L。
Described chemiluminescence preexciting liquid A is H2O2And HNO3Mixed liquor, wherein H2O2Mass fraction be 0.05-
5%, HNO3Concentration be 0.05-2.5 mol/L.
Described chemiluminescence exciting liquid B is Triton X-100 and NaOH mixed liquor, wherein Triton X-100's
Concentration is that 0.05-2.0 mol/L, NaOH concentration are 0.05-1.0 mol/L.
Described cleaning fluid is：PH 7.0-9.0, the Tris-HCl solution that concentration is 5.0-50.0 mmol/L, wherein containing
Concentration is 0.05-0.50 mol/L NaCl and 0.01-0.25% Tween-20.
The principle of the present invention is to be combined the high degree of specificity of antibody-antigene reaction with the high sensitivity that acridinium ester lights
Get up, using photon caused by acridinium ester catching reaction to detect production concentration.
The advantage of the invention is that combining magnetic microparticle chemiluminescence technology using competition law, the Rec DOPA in food is determined
Amine content.Acridinium ester has a clear superiority as the direct chemiluminescence of label, is mainly manifested in：Reaction need not be catalyzed
Agent, as long as alkaline environment can be carried out, be swift in response, can photon completely caused by catching reaction, background luminescence is low, signal to noise ratio
Height, disturbing factor is few, and reagent stability is good, and system is simple, and exciting liquid cost is low, and acridinium ester easily and protein bind, and is coupled
Photon yield is not reduced afterwards.
Embodiment
The present invention provides a kind of the magnetic microparticle chemiluminescence detection kit and preparation method of Ractopamine, to make this hair
Bright purpose, technical scheme and effect definitely, it is clear, the present invention is described in more detail below.
The present invention provides a kind of the magnetic microparticle chemiluminescence detection kit and preparation method of Ractopamine, wherein, this
The kit of the provided magnetic microparticle chemiluminescence method detection Ractopamine of invention, can take Ractopamine monoclonal
Antibody coupling magnetic particle, acridinium ester label Ractopamine antigen, Ractopamine antigen can also be taken to be coupled magnetic particle, a word used for translation
Pyridine ester marks ractopamine monoclonal antibody.The change that kit also includes Ractopamine calibration object, above-mentioned acridinium ester acts on
Learn luminous preexciting liquid A, chemiluminescence exciting liquid B and cleaning fluid.
Specifically, the kernel of magnetic bead of the present invention is ferroso-ferric oxide, described magnetic particle can directly with antibody or anti-
Original coupling, magnetic particle and Streptavidin can be also coupled, while use biotin labelled antibodies or antigen.It is solid before magnetic bead use
The endless bulk deposition of phase can influence accuracy, therefore should select good dispersion when selecting, and it is few to place magnetic bead number of uniting for a long time, sinks
Slow-footed magnetic bead drops.
Specifically, for the present invention when preparing magnetic particle suspension, the coupled antigen buffer solution is pH 5.0, concentration is
0.1 mol/L MES buffer solutions；The MES buffer solutions that coupled antibody buffer solution is pH 6.0, concentration is 0.1 mol/L.
Specifically, for the present invention when preparing magnetic particle suspension, the Block buffer is the buffer solution containing 1%BSA.
Specifically, calibration object of the present invention is with the Tris-HCl containing 1-3% BSA and 0.1-0.3% PC300
Buffer solution is matrix, adds the configuration of Ractopamine sterling and forms, calibration object form is liquid.
Specifically, chemiluminescence preexciting liquid A of the present invention is H2O2And HNO3Mixed liquor, wherein H2O2Quality
Fraction is 1.5%, HNO3Concentration be 0.1 mol/L.
Specifically, chemiluminescence exciting liquid B of the present invention is Triton X-100 and NaOH mixed liquor, wherein
Triton X-100 concentration is 0.1 mol/L, and NaOH concentration is 0.35 mol/L.
Specifically, cleaning fluid of the present invention is：PH 7.2, the Tris-HCl solution that concentration is 25 mmol/L, wherein containing
Concentration is 0.15 mol/L NaCl and 0.05% Tween-20.
Below by embodiment, the present invention is described in detail.
Embodiment 1：A kind of establishment of the magnetic microparticle chemiluminescence kit 1 of described detection Ractopamine and preparation side
Method, comprise the following steps：
1. the establishment of kit 1：
A kind of magnetic microparticle chemiluminescence kit for detecting Ractopamine is set up, it is contained following component：
The ractopamine monoclonal antibody of carboxyl magnetic particle coupling；
The Ractopamine antigen of acridinium ester label；
Ractopamine serial standards solution, concentration are respectively：0 μg/L、0.02 μg/L、0.1 μg/L、0.5 μg/L、
2.5 μ g/L, 12.5 μ g/L, its buffer solution are the Tris-HCl solution containing 1-3% BSA and 0.1-0.3% PC300；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
The Tris-HCl buffer solutions of cleaning fluid, the specially mmol/L of concentration 25（pH 7.2）, wherein containing the mol/L of concentration 0.15
NaCl and 0.05% Tween-20.
2. coupling has the preparation of the magnetic particle suspension of ractopamine monoclonal antibody
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds a certain amount of 0.1 mol/L MES buffer solutions, is vortexed mixed
It is even, it is placed on magnetic frame, standing 5 min makes magnetic particle be separated with liquid, supernatant discarding, washs 3 times, adds a certain amount of MES
（PH is 6.0）Buffer solution, it is vortexed.
（2）Add 15 μ L（15 μg）Ractopamine monoclonal antibody, be vortexed, revolving reaction pipe, incubation at room temperature 17
min。
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL to be vortexed, revolving reaction pipe, be incubated at room temperature 2 h.
（4）Supernatant is removed, adds a certain amount of cleaning buffer solution（TBS+0.05%Tween-20）, wash 3 times.
（5）Closed with the buffer solution containing 1%BSA, repeatedly closing 4 times, every time 10 min.By the magnetic particle suspension
It is placed in 2-8 DEG C of preservation.
3. prepared by the Ractopamine antigen liquid-phase reagent of acridinium ester label
（1）Purify Ractopamine：A certain amount of Ractopamine antigen is placed in bag filter, and bag filter is placed in not small
Dialysed in 1 L mark buffer solution, during which buffer solution is at least changed 3 times, last time dialysed overnight, and mark buffer solution is pH
10.1st, concentration is 0.1 mol/L Na2CO3-NaHCO3Buffer solution.
（2）1.7 mg acridinium ester NSP-DMAE-NHS is weighed, is dissolved in 447 μ L anhydrous dimethyl formamides DMF, matches somebody with somebody
Into 6.5 mmol/L NSP-DMAE-NHS DMF solutions.
（3）It will be placed in through the Ractopamine solution after dialysis in 500 μ L centrifuge tubes（Lucifuge is reacted）, add 200 μ
L marks buffer solution, then adds a certain amount of 6.5 mmol/L NSP-DMAE-NHS DMF solutions, acridinium ester and Rec DOPA
The molar ratio of amine is 9.7:1,1 h is reacted at room temperature, adds the μ L of 10 g/L lysines 100, is continued to react 15 min, is made mark
Remember reaction terminating.
（4）Label NSP-DMAE-NHS-Ag and free NSP-DMAE-NHS passes through Sephadex G-50 posts（1×
25cm）Separation, the PBS for being 0.1mol/L with purification buffer pH 6.3, concentration are balanced and are eluted chromatographic column.
（5）Protein peak is detected with chromatograph in separation process, the chemiluminescence intensity and 280nm for measuring efflux respectively are inhaled
Shading value.
（6）The eluent that shading value is high and absorbance is big is collected, adds 1% BSA（Volume）After dispense.
4. Ractopamine calibration object is prepared
Ractopamine sterling is configured to mark with the Tris-HCl buffer solutions containing 1-3% BSA and 0.1-0.3% PC300
Show calibration of the concentration for 0 μ g/L, 0.02 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 2.5 μ g/L, 12.5 μ g/L totally 6 concentration
Product Grad.
5. prepared by chemiluminescence exciting liquid A, B
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, wherein H2O2Mass fraction be 1.5%, HNO3It is dense
Spend for 0.1 mol/L, be distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
（2）Chemiluminescence exciting liquid B is made up of Triton X-100 and NaOH mixed liquor.Wherein, Triton X-100
Concentration be 0.1 mol/L, NaOH concentration is 0.35 mol/L, is distributed into 20 mL/ branch with brown bottle, 2-8 DEG C of preservation is standby
With.
Embodiment 2：A kind of establishment of the magnetic microparticle chemiluminescence kit 2 of described detection Ractopamine and preparation side
Method, comprise the following steps：
1. the establishment of kit 2：
A kind of magnetic microparticle chemiluminescence kit for detecting Ractopamine is set up, it is contained following component：
The Ractopamine antigen of carboxyl magnetic particle coupling；
The ractopamine monoclonal antibody of acridinium ester label；
Ractopamine serial standards solution, concentration are respectively：0 μg/L、0.02 μg/L、0.1 μg/L、0.5 μg/L、
2.5 μ g/L, 12.5 μ g/L, its buffer solution are the Tris-HCl solution containing 1-3% BSA and 0.1-0.3% PC300；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
The Tris-HCl buffer solutions of cleaning fluid, the specially mmol/L of concentration 25（pH 7.2）, wherein containing the mol/L of concentration 0.15
NaCl and 0.05% Tween-20.
2. coupling has the preparation of the magnetic particle suspension of Ractopamine antigen
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds a certain amount of 0.1 mol/L MES buffer solutions, is vortexed mixed
It is even, it is placed on magnetic frame, standing 5 min makes magnetic particle be separated with liquid, supernatant discarding, washs 3 times, adds a certain amount of MES
（PH is 5.0）Buffer solution, it is vortexed.
（2）Add 15 μ L（15 μg）Ractopamine antigen, be vortexed, revolving reaction pipe, be incubated at room temperature 17 min.
（3）Add the coupling reagent EDC that 10 μ L concentration are 10 mg/mL to be vortexed, revolving reaction pipe, be incubated at room temperature 2 h.
（4）Supernatant is removed, adds 200 μ L cleaning buffer solution（TBS+0.05% Tween-20）, wash 3 times.
（5）Closed with the buffer solution containing 1%BSA, repeatedly closing 4 times, every time 10 min.By the magnetic particle suspension
It is placed in 2-8 DEG C of preservation.
3. prepared by the ractopamine monoclonal antibody liquid-phase reagent of acridinium ester label
（1）Purify ractopamine monoclonal antibody：A certain amount of ractopamine monoclonal antibody is placed in bag filter, and
Bag filter is placed in the mark buffer solution not less than 1 L and dialysed, during which buffer solution is at least changed 3 times, is dialysed for the last time
Night, the Na that mark buffer solution is pH 10.1, concentration is 0.1 mol/L2CO3-NaHCO3Buffer solution.
（2）1.7 mg acridinium ester NSP-DMAE-NHS is weighed, is dissolved in 447 μ L anhydrous dimethyl formamides DMF, matches somebody with somebody
Into 6.5 mmol/L NSP-DMAE-NHS DMF solutions.
（3）It will be placed in through the ractopamine monoclonal antibody solution after dialysis in 500 μ L centrifuge tubes（Lucifuge is anti-
Should）, 200 μ L mark buffer solutions are added, then add a certain amount of 6.5 mmol/L NSP-DMAE-NHS DMF solutions, a word used for translation
Pyridine ester and the molar ratio of ractopamine monoclonal antibody are 7.4:1,1h is reacted at room temperature, adds 10 g/L lysines 100
μ L, continue to react 15 min, make mark reaction terminating.
（4）Label NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS passes through Sephadex G-50 posts（1×
25cm）Separation, balanced with purification buffer pH 6.3, the PBS that concentration is 0.1 mol/L and elute chromatographic column.
（5）Protein peak is detected with chromatograph in separation process, the chemiluminescence intensity and 280nm for measuring efflux respectively are inhaled
Shading value.
（6）The eluent that shading value is high and absorbance is big is collected, adds 1% BSA（Volume）After dispense.
4th, Ractopamine calibration object is prepared
Ractopamine sterling is configured to mark with the Tris-HCl buffer solutions containing 1-3% BSA and 0.1-0.3% PC300
Show calibration of the concentration for 0 μ g/L, 0.02 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 2.5 μ g/L, 12.5 μ g/L totally 6 concentration
Product Grad.
5. prepared by chemiluminescence exciting liquid A, B
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, H2O2Mass fraction be 1.5%, HNO3Concentration be
0.1 mol/L, 20 mL/ branch are distributed into brown bottle, 2-8 DEG C saves backup.
（2）Chemiluminescence exciting liquid B is made up of Triton X-100 and NaOH mixed liquor.Wherein, Triton X-100
Concentration be 0.1 mol/L, NaOH concentration is 0.35 mol/L, is distributed into 20 mL/ branch with brown bottle, 2-8 DEG C of preservation is standby
With.
Embodiment 3：The pre-treatment of sample
A. the processing of muscle, internal organ
The processing of muscle：The tissue samples after 2.0 g homogeneous are weighed into 50 mL centrifuge tubes；Add 4 mL, 2% NaCl and
0.2 mol/L HCl- methyl alcohol mixed liquors, vibrate 30 s；0.5 mL supernatants are taken (to keep away after 3000 r/min, room temperature centrifugation 5min
Open upper strata suspension) 35 mL, 0.5 mol/L NaOH solutions are added, add 0.5 mL, the phosphoric acid that concentration is 0.02 mol/L
Salt buffer, mix.
The processing of internal organ：The tissue samples after 2.0 g homogeneous are weighed into 50 mL centrifuge tubes；Add 4 mL, 2%NaCl with
And 0.2 mol/L HCl- methyl alcohol mixed liquor, vibrate 30 s；3000 r/min, room temperature take 0.5 mL supernatants after centrifuging 5 min
(avoiding upper strata suspension) adds 20 mL, 0.5 mol/L NaOH solutions, adds 0.5 mL, concentration is 0.02 mol/L's
Phosphate buffer, mix.
B. the processing of milk test sample solution
150 μ L fresh milks are taken in 500 μ L centrifuge tubes, 4 DEG C of 10 min of centrifugation（3000 r/min）, discard upper-layer fat.
Pipette the μ L of milk sample 25 after centrifugation to be placed in clean teat glass, add 950 μ L, concentration is 0.02 mol/L phosphoric acid
Salt buffer is diluted.
Embodiment 4：The step of being detected using the magnetic microparticle chemiluminescence detection kit of described Ractopamine
For：
（1）By μ L of sample to be tested 100, μ L of coupling magnetic particle suspension 150, the μ L of acridinium ester label 150, sequentially add anti-
Ying Guanzhong, vibration mix, 37 DEG C of 15 min of incubation.
（2）Separation cleaning 5 times.
（3）Reaction vessel after washing, which is fully vibrated, makes magnetic particle scatter.
（4）100 μ L chemiluminescence exciting liquid B are added after adding 100 μ L chemiluminescence preexciting liquid A, 1 s, measure it
Relative luminous intensity, the content of Ractopamine luminous intensity proportion relation corresponding thereto in sample.
Embodiment 5：The performance indications of kit
（1）The sensitivity of kit
20 retests are carried out to zero standard solution, the average value for taking zero standard solution to determine adds 3 times of standard deviation, i.e.,
For the sensitivity of this kit.Sensitivity of this kit to Ractopamine is 0.025 μ g/L.
（2）The specificity of kit
The competition medicine similar to Ractopamine structure or function：Clenbuterol, salbutamol, Terbutaline.By kit
Step operation, Ractopamine, Clenbuterol, salbutamol, Terbutaline are separately added into, suppression curve are made, according to linear
Equation calculates 50% inhibition concentration of each medicine.Cross reacting rate is IC of the antibody to Ractopamine50It is more to Rec with antibody
The IC of bar amine competitor50The ratio between percentage.As a result show：This kit has higher specificity to Ractopamine, right
With Ractopamine structure or intimate competition equal no cross reaction of medicine.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (10)
1. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine, it is characterised in that：Have including coupling
The magnetic particle suspension of ractopamine monoclonal antibody, calibration object, the Ractopamine antigen of acridinium ester label, chemiluminescence
Preexciting liquid A, chemiluminescence exciting liquid B and cleaning fluid.
2. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 1, its
It is characterised by：Described magnetic particle directly can be coupled with ractopamine monoclonal antibody, or by magnetic particle and Streptavidin
Coupling, while use biotin labeling ractopamine monoclonal antibody.
3. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 1, its
It is characterised by：Described chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
4. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 1, its
It is characterised by：Described acridinium ester label is Ractopamine antigen.
5. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 1, its
It is characterised by：Described calibration object is with the Tris-HCl buffer solutions containing 0.5-5.0% BSA and 0.1-0.5% PC300
For matrix, the calibration object solution that Ractopamine sterling configures the series concentration gradient formed is added.
6. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 1, its
It is characterised by：Described chemiluminescence preexciting liquid A is by H2O2And HNO3Mixed liquor composition, chemiluminescence exciting liquid B by
Triton X-100 and NaOH mixed liquor composition.
7. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine, it is characterised in that：Have including coupling
The magnetic particle suspension of Ractopamine antigen, calibration object, the ractopamine monoclonal antibody of acridinium ester label, chemiluminescence
Preexciting liquid A, chemiluminescence exciting liquid B and cleaning fluid.
8. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 7, its
It is characterised by：Described magnetic particle directly can be coupled with Ractopamine antigen, or magnetic particle and Streptavidin are coupled, together
Shi Caiyong biotin labeling Ractopamine antigens.
9. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 7, its
It is characterised by：Described chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
10. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of Ractopamine according to claim 7, its
It is characterised by：Described acridinium ester label is ractopamine monoclonal antibody.
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| CN110297092A (en) * | 2019-07-23 | 2019-10-01 | 山东绿都生物科技有限公司 | A kind of chemiluminescence detection kit of vomitoxin and preparation method thereof |
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