CN106568943A - Detection kit for ractopamine in food - Google Patents
Detection kit for ractopamine in food Download PDFInfo
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- CN106568943A CN106568943A CN201611027948.1A CN201611027948A CN106568943A CN 106568943 A CN106568943 A CN 106568943A CN 201611027948 A CN201611027948 A CN 201611027948A CN 106568943 A CN106568943 A CN 106568943A
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- G—PHYSICS
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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Abstract
The invention discloses a detection kit for ractopamine in food. The detection kit comprises (1) a ractopamine calibrator; (2) a ractopamine-amino magnetic bead connecter reagent; (3) enzyme conjugate; (4) a chemiluminescent substrate; and (5) a cleaning fluid. Ractopamine and a solid carrier are directly and stably connected; the stability of the connection between ractopamine and the solid carrier is guaranteed; at the same time, ractopamine molecules are fully exposed; at the same time, magnetic particles are added to increase the effective reaction area; and the ractopamine chemiluminescent immuno-detection kit has the advantages of higher sensitivity and shorter reaction time and is suitable for various luminescent detectors.
Description
Technical field
The present invention relates to technical field of food detection, the detection kit of Ractopamine in specifically a kind of food.
Background technology
Ractopamine (Ractopamin) belongs to beta-stimulants.Beta-stimulants are one kind of nutrient partition agent, are one
The phenylethanolamine analog derivative of class formation epinephrine similar with function and norepinephrine, it can improve lard type animal
Meat and the ratio of fat, reduce ketoboidies fat content, improve lean meat percentage, and can accelerate growth of animal, and be added in animal feeding
In material.Common β 2- analeptic has Clenbuterol, Ractopamine and albuterol etc..As China is to Clenbuterol（It is commonly called as
" clenbuterol hydrochloride "）The increasing of supervision, the use of Clenbuterol are gradually decreased, and anti-depressant uses of other β 2- gradually increases.By
Play the role of similar to Clenbuterol in Ractopamine, remain in animal tissue, so as to have a negative impact to human body, institute
Which is used as feed additive to forbid Ractopamine using China.
In terms of the detection method of Ractopamine, presently the most conventional method includes high performance liquid chromatography
（HPLC）, gas chromatography-mass spectrography（GC-MS）, liquid chromatograph mass spectrography（LC-MS）, capillary electrophoresis, enzyme linked immunological
Adsorption analyses method（Enzymelinkedimmunosorbentassay, ELISA）Etc. method.Although these methods can realize Lay
The accurate detection of gram dopamine, but its sensitivity, reaction efficiency have certain deficiency.
The content of the invention
It is an object of the invention to provide in the food that a kind of sensitivity is higher, the response time is short Ractopamine detection
Test kit.
For achieving the above object, the present invention provides following technical scheme：
The detection kit of Ractopamine in a kind of food, including：1）Ractopamine calibration object；2）Ractopamine-ammonia
Base magnetic bead junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.
The Concentraton gradient of described Ractopamine standard substance is 0ng/ml, 0.04ng/ml, 0.12ng/ml, 0.36ng/
ml、1.08ng/ml、3.24ng/ml。
The Main Ingredients and Appearance of described Ractopamine-amino magnetic bead junctional complex reagent is that Ractopamine is coupled amino magnetic bead
Junctional complex；The particle diameter of described amino magnetic bead is 2～4 μm, and surface active groups are amino（-NH2）.
The Main Ingredients and Appearance of described enzyme conjugates is the Ractopamine associated proteins of alkali phosphatase enzyme mark.
The Main Ingredients and Appearance of described chemical luminous substrate is (3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygens
Acyl)-phenyl -1,2- dioxane disodium salts).
In described food, the preparation method of the detection kit of Ractopamine, comprises the following steps：
1）Prepare Ractopamine calibration object；
2）Ractopamine is connected on amino magnetic bead and prepares junctional complex, with magnetic bead buffer solution dilution junctional complex to necessarily dense
Degree, prepares junctional complex reagent；
3）With enzyme labelling Ractopamine associated proteins, enzyme conjugates are prepared；
4）Prepare chemical luminous substrate；
5）Prepare cleanout fluid；
6）More than subpackage each reagent, constitutes finished product.
As further scheme of the invention：The preparation of described Ractopamine calibration object, comprises the following steps：
1）The preparation of calibration object buffer：Weigh 6g Tris alkali, 10g Sodium Chloride（NaCl）, 0.8g Proclin-300, add
900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrates；6g albumin rabbit serums are added, is settled to
Repetition measurement pH value after 1000ml, is allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of preservations；
2）Ractopamine sterling powder calibration object buffer solution, preparation are become into the concentrated solution of 10ng/ml, subsequently successively
Dilute 5 concentration, respectively：0.04ng/ml、0.12ng/ml、0.36ng/ml、1.08ng/ml、3.24ng/ml；Plus 0
Point, the calibration object Grad of totally 6 concentration.
As further scheme of the invention：The preparation of described Ractopamine-amino magnetic bead junctional complex reagent, including
Following steps：
1）Ractopamine 6mg is accurately weighed, is dissolved in pure water, final concentration of 3mg/ml obtains Ractopamine solution；
2）The phosphate buffer of the 30mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.8g、Na2HPO4·2H2O
3g, Sodium Chloride（NaCl）3.4g, adds 900ml purified water, pH value is adjusted to 7.0 with 2mol/L sodium citrates or sodium bicarbonate
± 0.1, repetition measurement pH value after 1000ml is settled to, is allowed in 7.0 ± 0.1,0.22 μm is filtered after 2～8 DEG C of preservations；
3）Prepare sulfosuccinimide 4- [N- the citraconic acids] -1- carboxylic hexamethylene of 6mg/ml（SMCC）, it is sub- with dimethyl
Sulfone（DMSO）Dissolving；
4）Sulfosuccinimide 4- [N- the citraconic acids] -1- carboxylics of the 6mg/ml of 85 μ L are added toward Ractopamine solution
Hexamethylene（SMCC）Activated, 20～22 DEG C are reacted 45 minutes, are fully mixed in course of reaction；
5）8mg amino magnetic beads are measured, amino magnetic bead is positioned on magnet stand and is stood 2 minutes, remove supernatant, with pH=7.0's
30mmol/L phosphate buffers wash three times and add phosphate buffer to preserve afterwards, make magnetic bead concentration be 25mg/ml；
6）Ractopamine solution after activation is added in the amino magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted
For 6mg/ml, 20～22 DEG C are reacted 40 minutes, are fully mixed in course of reaction；
7）Prepare magnetic bead buffer solution：Weigh 13g Tris alkali, 10g Sodium Chloride（NaCl）, 0.4ml tween 20s, 1.2g
Proclin-300, adds 900ml purified water, pH value is adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；Add 1.2g cheese
Plain sodium, is settled to repetition measurement pH value after 1000ml, is allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of preservations；
8）Supernatant is removed after the completion of reaction, three times is washed with 30mmol/L phosphate buffers and is added 1ml magnetic bead buffer solutions to enter afterwards
Row is preserved；During use, magnetic bead buffer solution is diluted to the working solution that concentration is 0.03mg/ml.
As further scheme of the invention：The preparation of described enzyme conjugates, comprises the following steps：
1）Alkali phosphatase 1.5mg is taken, concentration is adjusted to into 8mg/ml；
2）Weigh 6mg sulfosuccinimide 4- [N- citraconic acids] -1- carboxylic hexamethylene（SMCC）, use dimethyl sulfoxide
（DMSO）Being dissolved, final concentration of 16mg/ml, 5 μ L being added into alkali phosphatase, 20～22 DEG C are reacted 10 minutes；
3）Purification is carried out according to the difference of molecular weight to reactant mixture with molecular sieve chromatography, the alkaline phosphatase after activation is collected
Enzymatic solution；
4）Sterling Ractopamine associated proteins 0.6mg is taken, in adding the alkaline phosphatase buffer after activation, 2～8 DEG C of reactions
5 hours, finally the Ractopamine associated proteins of enzyme labelling are purified out with molecular sieve chromatography；
5）Enzyme conjugates buffer：Weigh 14g trishydroxymethylaminomethane（Tris）, 8g Sodium Chloride（NaCl）, 7g glycerol,
0.4g Proclin-300, add 900ml purified water, pH value are adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；Add 12g
Albumin rabbit serum, is settled to repetition measurement pH value after 1000ml, is allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of guarantors
Deposit；
6）By conjugate enzyme conjugates buffer be diluted to enzyme labelling Ractopamine associated proteins concentration be 3 μ g/ml i.e.
For working solution.
As further scheme of the invention：The preparation of described chemical luminous substrate, including：3- (2- spiral shells are weighed accurately
Rotation diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-phenyl -1,2- dioxane disodium salts（AMPPD）0.6g, trihydroxy methyl
Aminomethane（Tris）20g, Sodium Chloride（NaCl）80g, hexadecyltrimethylammonium chloride（CTAC）0.022g, purified water constant volume
To 1000ml, adjustment chemical luminous substrate solution ph is 9.4 ± 0.05.This chemical luminous substrate is for alkali phosphatase
Epidioxy ethane substrate, needs keep in dark place under the conditions of 2～8 DEG C, and the used time slowly mixes.
As further scheme of the invention：The preparation of described cleanout fluid, including：Weigh 1.2g trihydroxy methyl amino first
Alkane（Tris）, 8g Sodium Chloride（NaCl）, 0.8g tween 20s, add purified water about 900ml, mix to each reagent dissolve, use
2mol/L sodium citrate solutions adjust solution ph to 7.8 ± 0.1, add purified water and are settled to 1000ml, mix and treat after filtration
With.
The method detected using the detection kit of Ractopamine in described food, step is：
（1）40 μ L samples or calibration object are added in flat based tubes；
（2）40 μ L Magneto separate reagents are added in flat based tubes；
（3）50 μ L Ractopamine associated proteins alkaline phosphatase conjugates are added in flat based tubes；
（4）Flat based tubes are placed on vortex blending instrument and are mixed 30 seconds, be subsequently placed in 37 DEG C and react 25 minutes；
（5）Flat based tubes are placed on after 2 minutes being stood on magnet stand and outwell supernatant, pat dry, add 600 μ L of cleanout fluid, mix 20
Second；
（6）Repeat step（5）Twice；
（7）The flat based tubes for patting dry are put in immunity analysis instrument and are detected.
Compared with prior art, the invention has the beneficial effects as follows：The present invention is direct with solid phase carrier by Ractopamine
The method being stably connected with, can not only ensure the stability being connected with solid phase carrier but also can guarantee that Ractopamine molecule is obtained fully
Exposure, while also expanding effective affecting acreage by introducing magnetic particle, establishes that sensitivity is higher, the Lay that the response time is shorter
Gram dopamine chemiluminescence immune detection reagent kit, the test kit are applicable to various luminous detection instruments.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.Based in the present invention
Embodiment, the every other embodiment obtained under the premise of creative work is not made by those of ordinary skill in the art, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, the detection kit of Ractopamine in a kind of food, including：1）Ractopamine calibration object；
2）Ractopamine-amino magnetic bead junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.Ractopamine
The Concentraton gradient of standard substance is 0ng/ml, 0.04ng/ml, 0.12ng/ml, 0.36ng/ml, 1.08ng/ml, 3.24ng/ml.Lay
The Main Ingredients and Appearance of gram dopamine-amino magnetic bead junctional complex reagent is that Ractopamine is coupled amino magnetic bead junctional complex.Amino magnetic bead
Particle diameter be 2～4 μm, surface active groups are amino（-NH2）.The Main Ingredients and Appearance of enzyme conjugates is alkali phosphatase enzyme mark
Ractopamine associated proteins.The Main Ingredients and Appearance of chemical luminous substrate is (3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus
Oxygen acyl)-phenyl -1,2- dioxane disodium salts).
In described food, the preparation method of the detection kit of Ractopamine, comprises the following steps：
1）Ractopamine calibration object is prepared, including：The preparation of calibration object buffer：Weigh 6g Tris alkali, 10g Sodium Chloride
（NaCl）, 0.8g Proclin-300, add 900ml purified water, pH value is adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；
6g albumin rabbit serums are added, repetition measurement pH value after 1000ml is settled to, is allowed in 7.2 ± 0.1,0.22 μm is filtered after 2～8
DEG C preserve；By Ractopamine sterling powder calibration object buffer solution, preparation becomes the concentrated solution of 10ng/ml, subsequently according to
It is secondary to dilute 5 concentration, respectively：0.04ng/ml、0.12ng/ml、0.36ng/ml、1.08ng/ml、3.24ng/ml；Add
0 point, the calibration object Grad of totally 6 concentration；
2）Ractopamine is connected on amino magnetic bead and prepares junctional complex, with magnetic bead buffer solution dilution junctional complex to necessarily dense
Degree, prepares junctional complex reagent, including：Ractopamine 6mg is accurately weighed, is dissolved in pure water, final concentration of 3mg/ml is obtained
To Ractopamine solution；The phosphate buffer of the 30mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.8g、
Na2HPO4·2H2O 3g, Sodium Chloride（NaCl）3.4g, adds 900ml purified water, will with 2mol/L sodium citrates or sodium bicarbonate
PH value is adjusted to 7.0 ± 0.1, is settled to repetition measurement pH value after 1000ml, is allowed in 7.0 ± 0.1, and 0.22 μm is filtered after 2～8
DEG C preserve；Prepare sulfosuccinimide 4- [N- the citraconic acids] -1- carboxylic hexamethylene of 6mg/ml（SMCC）, it is sub- with dimethyl
Sulfone（DMSO）Dissolving；Sulfosuccinimide 4- [the N- methyl Malaysias of the 6mg/ml of 85 μ L are added toward Ractopamine solution
Acid] -1- carboxylic hexamethylene（SMCC）Activated, 20～22 DEG C are reacted 45 minutes, are fully mixed in course of reaction；Measure 8mg ammonia
Base magnetic bead, amino magnetic bead is positioned on magnet stand and stands 2 minutes, remove supernatant, with the 30mmol/L phosphate-buffered of pH=7.0
Liquid washs three times and adds phosphate buffer to preserve afterwards, makes magnetic bead concentration be 25mg/ml；By the Ractopamine solution after activation
In adding the amino magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted to into 6mg/ml, 20～22 DEG C are reacted 40 minutes, instead
Fully mix during answering；Prepare magnetic bead buffer solution：Weigh 13g Tris alkali, 10g Sodium Chloride（NaCl）, 0.4ml tween 20s,
1.2g Proclin-300, add 900ml purified water, pH value are adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；Add
1.2g casein sodiums, are settled to repetition measurement pH value after 1000ml, are allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of guarantors
Deposit；Supernatant is removed after the completion of reaction, three times is washed with 30mmol/L phosphate buffers and is added 1ml magnetic bead buffer solutions to be protected afterwards
Deposit；During use, magnetic bead buffer solution is diluted to the working solution that concentration is 0.03mg/ml；
3）With enzyme labelling Ractopamine associated proteins, enzyme conjugates are prepared, including：Alkali phosphatase 1.5mg is taken, concentration is adjusted
It is whole for 8mg/ml；Weigh 6mg sulfosuccinimide 4- [N- citraconic acids] -1- carboxylic hexamethylene（SMCC）, it is sub- with dimethyl
Sulfone（DMSO）Being dissolved, final concentration of 16mg/ml, 5 μ L being added into alkali phosphatase, 20～22 DEG C are reacted 10 minutes；With
Molecular sieve chromatography carries out purification according to the difference of molecular weight to reactant mixture, collects the alkaline phosphatase enzymatic solution after activation；
Sterling Ractopamine associated proteins 0.6mg is taken, in adding the alkaline phosphatase buffer after activation, 2～8 DEG C are reacted 5 hours,
Finally the Ractopamine associated proteins of enzyme labelling are purified out with molecular sieve chromatography；Enzyme conjugates buffer：Claim
Take 14g trishydroxymethylaminomethane（Tris）, 8g Sodium Chloride（NaCl）, 7g glycerol, 0.4g Proclin-300, add 900ml
Purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrates；12g albumin rabbit serums are added, 1000ml is settled to
Repetition measurement pH value, is allowed in 7.2 ± 0.1 afterwards, and 0.22 μm is filtered after 2～8 DEG C of preservations；By conjugate enzyme conjugates buffer
The Ractopamine associated proteins concentration for being diluted to enzyme labelling is working solution for 3 μ g/ml；
4）Chemical luminous substrate is prepared, including：3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-benzene is weighed accurately
Base -1,2- dioxane disodium salts（AMPPD）0.6g, trishydroxymethylaminomethane（Tris）20g, Sodium Chloride（NaCl）80g、
Hexadecyltrimethylammonium chloride（CTAC）0.022g, purified water are settled to 1000ml, adjust chemical luminous substrate solution ph
For 9.4 ± 0.05；This chemical luminous substrate is the epidioxy ethane substrate for alkali phosphatase, is needed in 2～8 DEG C of conditions
Under keep in dark place, the used time slowly mixes；
5）Cleanout fluid is prepared, including：Weigh 1.2g trishydroxymethylaminomethane（Tris）, 8g Sodium Chloride（NaCl）, 0.8g tweens-
20, add purified water about 900ml, mix to each reagent and dissolve, with 2mol/L sodium citrate solutions adjust solution ph to 7.8 ±
0.1, add purified water and be settled to 1000ml, mix after filtration stand-by；
6）More than subpackage each reagent, constitutes finished product.
The method detected using the detection kit of Ractopamine in described food, step is：
（1）40 μ L samples or calibration object are added in flat based tubes；
（2）40 μ L Magneto separate reagents are added in flat based tubes；
（3）50 μ L Ractopamine associated proteins alkaline phosphatase conjugates are added in flat based tubes；
（4）Flat based tubes are placed on vortex blending instrument and are mixed 30 seconds, be subsequently placed in 37 DEG C and react 25 minutes；
（5）Flat based tubes are placed on after 2 minutes being stood on magnet stand and outwell supernatant, pat dry, add 600 μ L of cleanout fluid, mix 20
Second；
（6）Repeat step（5）Twice；
（7）The flat based tubes for patting dry are put in immunity analysis instrument and are detected.
The judgement of testing result：By the meansigma methodss of the standard substance for being obtained and sample luminous intensity values divided by first standard
（0 standard）Luminous intensity values be multiplied by 100 again, with suppression ratio as vertical coordinate, the logarithm of Ractopamine concentration is that abscissa is made
Standard curve, the concentration of each sample can be read from standard curve.
Relative luminous intensity（%）=RLU/RLU0, RLU is the luminous intensity that standard substance or sample solution are determined, RLU0It is
It is blank（Concentration is 0 standard solution）Luminous intensity values.
The accuracy and precision of test kit
Accuracy refers to the matching degree between measured value and true value, and the conventional response rate of test kit accuracy is represented.Precision also known as
Repeatability, the conventional coefficient of variation are represented.
According to sample-pretreating method, with the Ractopamine of two concentration of 0.1ng/ml, 0.2ng/ml respectively to milk,
Powdered milk sample is added recovery, and every kind of sample each concentration mensuration 5 is parallel, is measured with three batches of test kits, calculates sample
The response rate and precision of product.Experimental result shows, in milk sample the TIANZHU XINGNAO Capsul scope of Ractopamine 87.1～
101.2%, in powdered milk sample, the TIANZHU XINGNAO Capsul scope of Ractopamine is 82.9～98.5%.Within-run and between-run analysis coefficient is equal
Less than 10%.
The specificity of test kit
Using Ractopamine as standard, if the cross reacting rate of Ractopamine is 100%, grind for antibody cross reaction
The medicine studied carefully is and Ractopamine structure or intimate medicine：Clenbuterol, albuterol.By test kit step
Operation, but the competitor for adding is respectively Ractopamine, clenbuterol, albuterol, makes suppression curve, according to linear side
Journey calculates each 50% inhibition concentration of competitor（IC50）.Cross reacting rate（%CR）As IC of the antibody to Ractopamine50With it is anti-
IC of the body to Ractopamine competitor50Ratio percent.As a result show, clenbuterol, the cross reacting rate of albuterol
＜ 1%, illustrates that the test kit has good specificity.
The method that the present invention is directly stably connected with by Ractopamine and solid phase carrier, can both ensure to connect with solid phase carrier
The stability for connecing can guarantee that Ractopamine molecule is fully exposed again, while also expanding effective by introducing magnetic particle
Response area, establishes the Ractopamine chemiluminescence immune detection reagent kit that sensitivity is higher, the response time is shorter, the examination
Agent box is applicable to various luminous detection instruments.
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 spirit or essential attributes without departing substantially from the present 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 is required rather than described above is limited, 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 this specification is been described by according to embodiment, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of description is only that those skilled in the art should for clarity
Using description as an entirety, the technical scheme in each embodiment can also Jing it is appropriately combined, form those skilled in the art
Understandable other embodiment.
Claims (10)
1. in a kind of food Ractopamine detection kit, it is characterised in that include：1）Ractopamine calibration object；2）
Ractopamine-amino magnetic bead junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.
2. in food according to claim 1 Ractopamine detection kit, it is characterised in that described Rec is more
The Concentraton gradient of bar amine standard substance is 0ng/ml, 0.04ng/ml, 0.12ng/ml, 0.36ng/ml, 1.08ng/ml, 3.24ng/
ml。
3. in food according to claim 1 Ractopamine detection kit, it is characterised in that described Rec is more
The Main Ingredients and Appearance of bar amine-amino magnetic bead junctional complex reagent is that Ractopamine is coupled amino magnetic bead junctional complex；Described amino magnetic
The particle diameter of pearl is 2～4 μm, and surface active groups are amino（-NH2）.
4. in food according to claim 1 Ractopamine detection kit, it is characterised in that described enzyme is combined
The Main Ingredients and Appearance of thing is the Ractopamine associated proteins of alkali phosphatase enzyme mark.
5. in food according to claim 1 Ractopamine detection kit, it is characterised in that described chemistry send out
The Main Ingredients and Appearance of light substrate is (3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-phenyl -1,2- dioxanes
Disodium salt).
6. the preparation method according to the detection kit of Ractopamine in the arbitrary described food of Claims 1 to 5, its feature
It is to comprise the following steps：
1）Prepare Ractopamine calibration object；
2）Ractopamine is connected on amino magnetic bead and prepares junctional complex, with magnetic bead buffer solution dilution junctional complex to necessarily dense
Degree, prepares junctional complex reagent；
3）With enzyme labelling Ractopamine associated proteins, enzyme conjugates are prepared；
4）Prepare chemical luminous substrate；
5）Prepare cleanout fluid；
6）More than subpackage each reagent, constitutes finished product.
7. in food according to claim 6 the detection kit of Ractopamine preparation method, it is characterised in that institute
The preparation of the Ractopamine stated-amino magnetic bead junctional complex reagent, comprises the following steps：
1）Ractopamine 6mg is accurately weighed, is dissolved in pure water, final concentration of 3mg/ml obtains Ractopamine solution；
2）The phosphate buffer of the 30mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.8g、Na2HPO4·2H2O
3g, Sodium Chloride 3.4g, add 900ml purified water, pH value are adjusted to 7.0 ± 0.1 with 2mol/L sodium citrates or sodium bicarbonate,
Repetition measurement pH value after 1000ml is settled to, is allowed in 7.0 ± 0.1,0.22 μm is filtered after 2～8 DEG C of preservations；
3）Sulfosuccinimide 4- [N- the citraconic acids] -1- carboxylic hexamethylene of 6mg/ml is prepared, dmso solution is used；
4）Sulfosuccinimide 4- [N- the citraconic acids] -1- carboxylics of the 6mg/ml of 85 μ L are added toward Ractopamine solution
Hexamethylene is activated, and 20～22 DEG C are reacted 45 minutes, are fully mixed in course of reaction；
5）8mg amino magnetic beads are measured, amino magnetic bead is positioned on magnet stand and is stood 2 minutes, remove supernatant, with pH=7.0's
30mmol/L phosphate buffers wash three times and add phosphate buffer to preserve afterwards, make magnetic bead concentration be 25mg/ml；
6）Ractopamine solution after activation is added in the amino magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted
For 6mg/ml, 20～22 DEG C are reacted 40 minutes, are fully mixed in course of reaction；
7）Prepare magnetic bead buffer solution：13g Tris alkali, 10g Sodium Chloride, 0.4ml tween 20s, 1.2g Proclin-300 are weighed,
900ml purified water is added, pH value is adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；1.2g casein sodiums are added, is settled to
Repetition measurement pH value after 1000ml, is allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of preservations；
8）Supernatant is removed after the completion of reaction, three times is washed with 30mmol/L phosphate buffers and is added 1ml magnetic bead buffer solutions to enter afterwards
Row is preserved；During use, magnetic bead buffer solution is diluted to the working solution that concentration is 0.03mg/ml.
8. in food according to claim 6 the detection kit of Ractopamine preparation method, it is characterised in that institute
The preparation of the enzyme conjugates stated, comprises the following steps：
1）Alkali phosphatase 1.5mg is taken, concentration is adjusted to into 8mg/ml；
2）6mg sulfosuccinimide 4- [N- citraconic acids] -1- carboxylic hexamethylene is weighed, is dissolved with dimethyl sulfoxide,
Final concentration of 16mg/ml, adds 5 μ L into alkali phosphatase, and 20～22 DEG C are reacted 10 minutes；
3）Purification is carried out according to the difference of molecular weight to reactant mixture with molecular sieve chromatography, the alkaline phosphatase after activation is collected
Enzymatic solution；
4）Sterling Ractopamine associated proteins 0.6mg is taken, in adding the alkaline phosphatase buffer after activation, 2～8 DEG C of reactions
5 hours, finally the Ractopamine associated proteins of enzyme labelling are purified out with molecular sieve chromatography；
5）Enzyme conjugates buffer：Weigh 14g trishydroxymethylaminomethane, 8g Sodium Chloride, 7g glycerol, 0.4g
Proclin-300, adds 900ml purified water, pH value is adjusted to 7.2 ± 0.1 with 2mol/L sodium citrates；Add 12g Sanguis Leporis seu oryctolagi
Pure albumen, is settled to repetition measurement pH value after 1000ml, is allowed in 7.2 ± 0.1, and 0.22 μm is filtered after 2～8 DEG C of preservations；
6）By conjugate enzyme conjugates buffer be diluted to enzyme labelling Ractopamine associated proteins concentration be 3 μ g/ml i.e.
For working solution.
9. in food according to claim 6 the detection kit of Ractopamine preparation method, it is characterised in that institute
The preparation of the chemical luminous substrate stated, including：3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyls)-benzene is weighed accurately
Base -1,2- dioxane disodium salt 0.6g, trishydroxymethylaminomethane 20g, Sodium Chloride 80g, cetyl trimethyl chlorination
Ammonium 0.022g, purified water are settled to 1000ml, and adjustment chemical luminous substrate solution ph is 9.4 ± 0.05.
10. in food according to claim 6 the detection kit of Ractopamine preparation method, it is characterised in that
The preparation of described cleanout fluid, including：1.2g trishydroxymethylaminomethane, 8g Sodium Chloride, 0.8g tween 20s are weighed, is added pure
Change water 900ml, mix to each reagent and dissolve, solution ph is adjusted to 7.8 ± 0.1 with 2mol/L sodium citrate solutions, added pure
Change water and be settled to 1000ml, mix after filtration stand-by.
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| CN201611027948.1A CN106568943A (en) | 2016-11-22 | 2016-11-22 | Detection kit for ractopamine in food |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108627643A (en) * | 2018-06-24 | 2018-10-09 | 沭阳康源泰博生物科技有限公司 | A kind of Ractopamine Sample pretreatment kit based on immunomagnetic beads |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928410A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting ractopamin, and applications thereof |
| CN104198721A (en) * | 2014-07-28 | 2014-12-10 | 北京润诺思医疗科技有限公司 | Preparation and application of Golgi protein 73 (GP73) antigen silicon-based magnetic bead conjugate |
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2016
- 2016-11-22 CN CN201611027948.1A patent/CN106568943A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928410A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting ractopamin, and applications thereof |
| CN104198721A (en) * | 2014-07-28 | 2014-12-10 | 北京润诺思医疗科技有限公司 | Preparation and application of Golgi protein 73 (GP73) antigen silicon-based magnetic bead conjugate |
Non-Patent Citations (1)
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| 宋宏新: "《食品免疫学》", 30 April 2009 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108627643A (en) * | 2018-06-24 | 2018-10-09 | 沭阳康源泰博生物科技有限公司 | A kind of Ractopamine Sample pretreatment kit based on immunomagnetic beads |
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