CN107884567A - A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit - Google Patents
A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit Download PDFInfo
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- CN107884567A CN107884567A CN201711296696.7A CN201711296696A CN107884567A CN 107884567 A CN107884567 A CN 107884567A CN 201711296696 A CN201711296696 A CN 201711296696A CN 107884567 A CN107884567 A CN 107884567A
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- magnetic bead
- cbl
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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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of magnetic bead time-resolved fluoroimmunoassay kit for quantitatively detecting Clenbuterol, the kit is coated with magnetic bead by BSA CBL, fluorescent material marks CBL antibody, cleaning solution, enhancing liquid and Clenbuterol standard solution form.The sensitivity of this kit can reach 0.1ppb, and precision is high, with Clenbuterol 26S Proteasome Structure and Function analog no cross reaction.The Clenbuterol magnetic bead time-resolved fluoroimmunoassay kit of the present invention that quantitatively detects has high sensitivity, more accurately advantage compared with the methods of colloidal gold immunity chromatography, fluorescence immune chromatography method；It is short compared to HPLC, GC/MS, ELISA detection time, it is cheap, great amount of samples Site Detection can be realized.
Description
Technical field
The present invention relates to field of detection of food safety, and specifically, the present invention relates to a kind of magnetic bead time-resolved fluorescence to exempt from
Epidemic disease quantitatively detects the kit of Clenbuterol.
Background technology
Clenbuterol（Clenbuterol, CBL）It is a kind of receptor,β activator, mainly with hydrochloride form
In the presence of adding Clenbuterol in feed, can promote growth of animal, suppress fat deposition, improve protein content, increase ketoboidies
Lean meat percentage, improve the effect such as meat.Therefore, in recent years under the driving of economic interests, criminal uses a large amount of Clenbuterols
In animal husbandry and aquaculture.Human body, which takes in excessive CBL, can cause a series of adverse reactions, mainly have muscular tremor, heartbeat and
Accelerated breathing etc., serious entail dangers to life.In consideration of it, many countries have forbidden adding Clenbuterol as feed and feed
Agent is added to use, the Ministry of Agriculture of China was also clearly included in 2002《Forbid the medicine used in feed and animal drinking water
Kind catalogue》, and provide that CBL must not be detected in animal food.
The detection method of Clenbuterol mainly has 5 kinds at present：Gas chromatography-mass spectrometry（GC/MS）, high-efficient liquid phase color
Spectrometry（HPLC）, enzyme connects immunoassay, colloidal gold immunity chromatography, fluorescence immune chromatography method；GC/MS can be used for feed, blood
The analysis of clenobuterol hydrochloride, detection are limited to 0.5 ppb in liquid, tissue, and compared with high performance liquid chromatography, detection sensitivity is more
Height, false positive rate are lower.Therefore, gas chromatography-mass spectrometry is set to detection hydrochloric acid gram by the country such as China, European Union and U.S.
Lun Teluo authenticity method.HPLC has the characteristics of detection accuracy is high, false positive rate is low, and its lowest detection is limited to 1 ~
15ppb, the domestic half authenticity method using high performance liquid chromatography as detection clenobuterol hydrochloride residue.Foregoing two kinds of inspections
Survey method shortcoming is that detection process is loaded down with trivial details, detection time length, needs expensive instrument, is difficult to operate, is expensive.ELISA
Have the advantages that detection accurate, high sensitivity, instrument and equipment are simple, but detection time it is long, it is cumbersome can not realize it is live fast
Speed detection.Collaurum and fluorescence immune chromatography reagent have easy to operate, quick, reagent safety, do not need specific apparatus and set
The advantages that standby, but it is not high the degree of accuracy to be present, the shortcomings such as sensitivity is low.
The content of the invention
It is an object of the invention to overcome above-mentioned technological deficiency, there is provided a kind of cost is cheap, simple to operate, accurate, sensitive
Magnetic bead time-resolved fluoroimmunoassay quantitatively detect Clenbuterol kit.
To achieve the above object, present invention employs following technical scheme：
A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, including BSA-CBL coatings magnetic bead, fluorescence
Matter mark CBL antibody, cleaning solution, enhancing liquid and Clenbuterol standard solution.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described BSA-CBL bags
It is the super-paramagnetic bead of 1 ~ 3 μm of diameter and the covalent coupling thing of albumen with modified with functional group by magnetic bead, the magnetic bead is carboxyl
Magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic beads, streptavidin magnetic bead, albumin A magnetic bead, Protein G
One or more in magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic bead.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described fluorescent material is
Group of the lanthanides huge legendary turtle and thing.Described fluorescent material is by europium（Eu3+）, samarium（Sm3+）, terbium（Tb3+）Or dysprosium（Dy3+）Formed Deng rare earth element
Chelate.The excitation wavelength of the fluorescent material is 300 ~ 350nm, and launch wavelength is 500 ~ 650nm.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described washing formula of liquid
For：The 0.05M Tris-Hcl buffer solutions of 0 .8 ~ 1 .2% Tween-20,0.05% Proclin300, pH 7.2 ~ 7.5.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described enhancing formula of liquid
For：0.03% sodium acetate, 0.0001 ~ 0.0005% β-NTA, 0.0024% TOPO, 0.08% acetic acid, 0.1% absolute ethyl alcohol, 0.05%
Triton X-100 the aqueous solution.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described Clenbuterol mark
Totally 6 bottles of quasi- product solution, concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described BSA-CBL bags
By the preparation method of magnetic bead：Carboxyl magnetic bead is put into centrifuge tube, centrifuge tube is placed in magnetic frame, is carried out using magnetic frame
The separation of magnetic bead and buffer solution, with 0.05M pH6.0 2-（N- morpholines）Ethyl sulfonic acid washing magnetic bead 3 ~ 5 times；To above-mentioned washing
0.05M pH6.0 2- is added in good magnetic bead（N- morpholines）Ethyl sulfonic acid, add 30 μ L 10mg/mL 1- (3- dimethylaminos
Propyl group) -3- ethyl carbodiimide hydrochlorides solution and 30 μ L 50mg/ml n-hydroxysuccinimides, room temperature concussion reaction 0.5 ~ 1
Hour；The magnetic bead activated is placed on magnetic frame, abandons supernatant, collects the magnetic bead activated.BSA-CBL is added, room temperature is lasting
Rotation is incubated 30 ~ 240 minutes；Magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads；Addition contains 1%BSA 0.05M
PH 8.0 Tris-HCl buffer solutions, magnetic bead is resuspended, closing is not coupled BSA-CBL activated carboxyl site, reacts 30 ~ 60min；
Magnetic bead is placed on magnetic frame, abandons supernatant, 100 μ L immunomagnetic beadses is added and preserves liquid.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described immunomagnetic beads is protected
Liquid storage is 5% sucrose containing 5%BSA, the 0.05M pH 8.0 of 0.1%Tween-20 and 0.1% polyvinylpyrrolidone Tris-HCl
Buffer solution.
In above-mentioned magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, described fluorescent material mark
Note CBL antibody europium labeling antibody preparation method be：CBL monoclonal antibodies are placed in 30KD ultra-filtration centrifuge tubes, centrifuges, discards filter
Liquid；Mark buffer solution is added, centrifugation, discards filtrate, repeats this operation 4 ~ 5 times；Centrifuge tube filter membrane is inverted, centrifuged, is collected dense
The antibody of contracting, mark buffer solution is added, is stood, then centrifuge tube filter membrane is inverted, centrifuged, collect the antibody of concentration；According to quality
Than CBL monoclonal antibody：Europium chelate DTTA-Eu=5:1 ratio fully mixes, and is put into rotary incubator, reacts at room temperature 16-
20 hours；Purify europium mark CBL antibody with SepHadex TM G-50 sephadex columns, then add europium labeling antibody preservative agent,
Make BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, store for future use；
Or the preparation method of the samarium labeling antibody of described fluorescent material mark CBL antibody is：CBL monoclonal antibodies are taken out to be placed in
30KD ultra-filtration centrifuge tubes, centrifugation, discard filtrate；Mark buffer solution is added, centrifugation, discards filtrate；Repeat this operation 4 ~ 5 times；Will
Centrifuge tube filter membrane inverts, centrifugation, collects the antibody of concentration；Compare antibody according to quality：Samarium chelate=4:1 ratio fully mixes,
Rotary incubator is put into, is reacted at room temperature 16 ~ 20 hours；Purify samarium mark CBL with SepHadex TM G-50 sephadex columns to resist
Body, samarium labeling antibody preservative agent is then added, makes BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, storage
It is standby.
The Cleaning Principle of detection Clenbuterol kit of the present invention is competition law, is pre-installed in the reacting hole of reagent strip
There are a BSA-CBL immunomagnetic beadses, during test, first by sample to be tested（Urine or extract solution）Or standard items are added to the reacting hole of reagent
In, add the clenbuterol monoclonal antibody that fluorescent material marks.By incubation at room temperature, Clenbuterol in sample to be tested with
The Clenbuterol monoclonal antibody of the coated BSA-CBL competition bindings fluorescent material mark of magnetic bead.After washing, do not combined with labelled antibody
Testing sample in Clenbuterol washed away, add enhancing liquid, in the presence of exciting light, fluorescent material launch a standing wave
Long optical signal, identify that CBL is more in sample by Immunofluorescence test instrument, the fluorescent material that the coated BSA-CBL of magnetic bead is combined
The Clenbuterol monoclonal antibody of mark is fewer, due in fluorescence signal intensity and sample Clenbuterol concentration inversely, by gram human relations
Special sieve concentration and fluorescence signal value fitted dose-response curve, you can obtain the dense of Clenbuterol in unknown sample by this measured value
Degree.
Compared with prior art, the present invention has the advantages that：
（1）The strong magnetic bead of coating uniform particle sizes, magnetic replaces the traditional reaction of elisa plate formula and the physical absorption of chromatography reaction
Mode, because there is magnetic bead bigger bonded area fully to be reacted with that can disperse in the liquid phase, detection range is greatly improved, is shortened
In the reaction time, sensitivity is improved, is expected to play a significant role to the residue detection of Clenbuterol in animal-derived food.Due to magnetic bead
It is covalent coupling with antigen or antibody, overcomes the unstability of physical absorption, therefore the immunomagnetic beads holding time is long and more steady
It is fixed.
（2）Using Time-resolved fluoroimmunoassay, using lanthanide chelate europium, terbium, samarium, dysprosium as label,
It has wider excitation spectrum, narrower emission spectrum, advantageously reduces background, improves sensitivity；Ultraviolet excitation have compared with
High quantum production rate, larger Stokes displacements, the spectrum weight for avoiding excitation spectrum and fluorescence emission spectrum and bio-matrix from launching
Close, the advantages that fluorescence decay time is long, it is more wider than conventional fluorescent material detection range, specific more preferable.
（3）Supporting full-automatic detecting instrument, realizes live automation mechanized operation, can detect one or more parts sample simultaneously, behaviour
Make easy to be quick（Result can be gone out within 20 minutes）, it is cheap.
Brief description of the drawings
Fig. 1 is that magnetic bead time-resolved fluoroimmunoassay is quantitatively detected Clenbuterol kit and detected using reagent strip, is tried
The schematic top plan view of agent bar structure.
Fig. 2 is that magnetic bead time-resolved fluoroimmunoassay is quantitatively detected Clenbuterol kit and detected using reagent strip, is tried
The schematic diagram of agent bar structure.
1st, instrument connection 1,2, instrument connection 2,3, label Isosorbide-5-Nitrae, label 2,5, label dilution, 6, washing lotion, 7, washing lotion,
8th, Sample dilution 1,9, Sample dilution 2,12, enhancing liquid, 10,11,13 be prepared hole.
Embodiment
The BSA-CBL coating magnetic beads of the present invention are the super-paramagnetic bead and albumen of 1 ~ 3 μm of the diameter with modified with functional group
Covalent coupling thing, wherein used magnetic bead has carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic
Pearl, streptavidin magnetic bead, albumin A magnetic bead, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic bead etc. are therein
It is one or more of.
Fluorescent material of the present invention is group of the lanthanides huge legendary turtle and thing, mainly there is europium（Eu3+）, samarium（Sm3+）, terbium（Tb3+）, dysprosium
（Dy3+）Deng the chelate of rare earth element composition.
The monoclonal antibody that CBL antibody employed in the present invention is prepared for conventional monoclonal antibody technology, used BSA-CBL
It is to synthesize to obtain using conventional chemical processes.
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
Embodiment 1
Magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, including BSA-CBL coatings magnetic bead, fluorescent material mark
Remember CBL antibody, cleaning solution, enhancing liquid and Clenbuterol standard solution.
Kit is detected using reagent strip, reagent strip is by instrument connection 1（BSA-CBL is coated with carboxyl magnetic bead）, mark
Remember thing hole 3（Europium mark CBL antibody）, washing lotion hole 6 with 7, enhancing fluid apertures 12 forms；The structure of reagent strip is as depicted in figs. 1 and 2.
The preparation process that the magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit is as follows, wherein
（1）BSA-CBL is coated with the preparation of magnetic bead：
Wash magnetic bead
1mg 1 μm of carboxyl magnetic bead of diameter is drawn into 1.5ml centrifuge tubes, centrifuge tube is placed in magnetic frame, utilizes magnetic frame
The separation of magnetic bead and buffer solution is carried out, with 1ml 0.05M pH6.0 2-（N- morpholines）Ethyl sulfonic acid（MES）Wash magnetic bead 3 ~ 5
It is secondary.
Magnetic bead activates
1ml 0.05M pH6.0 MES is added in the magnetic bead good to above-mentioned washing, adds 30 μ L 10mg/mL 1- (3- diformazans
Aminopropyl) -3- ethyl carbodiimide hydrochloride solution（EDC）With 30 μ L 50mg/ml n-hydroxysuccinimides（NHS）, room
Warm concussion reaction 0.5 ~ 1 hour.
BSA-CBL and magnetic bead coupling
The good magnetic bead of above-mentioned activation is placed on magnetic frame, abandons supernatant, collects the magnetic bead activated.Add 0.05 ~ 1mg BSA-
CBL, room temperature persistently rotate incubation 30 ~ 240 minutes, and the reaction time depends on the dentate and concentration of magnetic bead.
Closing
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.Addition contains 1%BSA 0.05M pH's 8.0
Tris-HCl buffer solutions, magnetic bead is resuspended, closing is not coupled BSA-CBL activated carboxyl site, reacts 30 ~ 60min.
Storage
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, is added 100 μ L immunomagnetic beadses and is preserved liquid.
Described preservation liquid is containing 5%（w/v）BSA, 5%（w/v）Sucrose, 0.1%（v/v）Tween-20 and 0.1%（w/
v）The 0.05M pH 8.0 of polyvinylpyrrolidone Tris-HCl buffer solutions.
The above-mentioned immunomagnetic beads prepared is diluted, makes BSA-CBL final concentration of 0.0025 ~ 0.0080g/L, packing is extremely
The hole of reagent strip the 1st（Instrument connection 1）In.
（2）The preparation method of europium labeling antibody：
1. the synthesis of europium labelled antibody
A. 0.5mg CBL monoclonal antibodies are taken out and are placed in 30KD ultra-filtration centrifuge tubes, 8000rpm centrifugation 6min, discard filtrate.
B. mark buffer solution is added（0.1M pH9.0 carbonate buffer solutions）200 μ L, 8000rpm centrifugation 6min, discard filter
Liquid.Repeat this operation 4 ~ 5 times.
C. centrifuge tube filter membrane is inverted, 3000rpm centrifugation 6min, collects the antibody of concentration.Add 100 μ L mark bufferings
Liquid, 3 ~ 5min is stood, then centrifuge tube filter membrane is inverted, 3000rpm centrifuges 6min, collects the antibody of concentration.
D. antibody is compared according to quality：Europium chelate DTPA-Eu=5:1 ratio fully mixes, and is put into rotary incubator, room
Temperature reaction 16 ~ 20 hours.
2. the purifying of europium labelled antibody
Purify europium mark CBL antibody with SepHadex TM G-50 sephadex columns, then add europium labeling antibody preservative agent（10%
（w/v）BSA+5%（w/v）Proclin300）, make BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, 4
DEG C store for future use.
The above-mentioned europium mark CBL antibody prepared is diluted by a certain percentage, make europium mark CBL final concentration of 0.005 ~
0.010g/L, dispense to the hole of reagent strip the 3rd（Label 1）In.
The excitation wavelength of described fluorescent material europium is 340nm, launch wavelength 615nm.
（3）The preparation method of cleaning solution：
Prepare containing the .2% of 0 .8 ~ 1（v/v）Tween-20,0.05%（v/v）The 0.05M of Proclin300, pH 7.2 ~ 7.5
Tris-Hcl buffer solutions, the solution prepared is dispensed into washing lotion hole 6 and 7 with 900 μ L/ holes.
（4）Strengthen the preparation method of liquid：
Preparation contains 0.03%（w/v）Sodium acetate, 0.0001 ~ 0.0005%（w/v）β-NTA、0.0024% （w/v）TOPO 、
0.08%（v/v）Acetic acid, 0.1%（v/v）Absolute ethyl alcohol, 0.05%（v/v）Triton X-100 the aqueous solution.By what is prepared
Solution is dispensed to the hole of reagent strip the 12nd with 300 μ L/ holes（Strengthen fluid apertures）.
（5）Clenbuterol standard solution preparation method：
Totally 6 bottles of described Clenbuterol standard solution, concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7
μg/L、8.1μg/L。
Embodiment 2
Magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, including BSA-CBL coatings magnetic bead, fluorescent material mark
Remember CBL antibody, cleaning solution, enhancing liquid and Clenbuterol standard solution.
Kit is detected using reagent strip, reagent strip is by the 2nd hole（Instrument connection 2）（BSA-CBL is coated with amino magnetic
Pearl）, the 4th hole（Label hole 4）（Samarium mark CBL antibody）, washing fluid apertures 6 and 7, enhancing fluid apertures form, as depicted in figs. 1 and 2.
The preparation process that the magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit is as follows, wherein
（1）BSA-CBL is coated with the preparation of magnetic bead：
1. wash magnetic bead
2 μm of amino magnetic beads of 1mg are drawn into 1.5ml centrifuge tubes, centrifuge tube is placed in magnetic frame, carried out using magnetic frame
The separation of magnetic bead and buffer solution, magnetic bead is washed 3 ~ 5 times with 1ml 0.01M pH6.0 pyridine.
Magnetic bead activates
1ml 0.01M pH6.0 pyridine is added in the good magnetic bead of above-mentioned washing, magnetic bead is resuspended, adds 100 μ L 25% penta 2
Aldehyde solution, room temperature concussion reaction 2 ~ 3 hours.
BSA-CBL and magnetic bead coupling
The good magnetic bead of above-mentioned activation is placed on magnetic frame, abandons supernatant, collects the magnetic bead activated.Add 1ml pH10.0 carbonic acid
Salt buffer, magnetic bead is resuspended, adds 0.05 ~ 1mg BSA-CBL, room temperature persistently rotates incubation 16 ~ 20 hours, and the reaction time takes
Certainly in dentate and concentration.
Closing
It is subsequently placed on magnetic frame, abandons supernatant, collects immunomagnetic beads.0.1M pH 8.0 glycine buffer is added, closing is not
BSA-CBL activation amino sites are coupled, react 30 ~ 60min.
Storage
Above-mentioned magnetic bead is placed on magnetic frame, abandons supernatant, is added 100 μ L immunomagnetic beadses and is preserved liquid.
Described preservation liquid is 5% trehalose and 0.1% Tween-20 0.05M pH 8.0 Tris- containing 8% BSA
HCl buffer solutions.
The above-mentioned immunomagnetic beads prepared is diluted by a certain percentage, dispenses BSA-CBL final concentration of 0.005g/L
To the hole of reagent strip the 2nd（Instrument connection 2）In.
（2）The preparation method of samarium labeling antibody is as follows：
1. the synthesis of samarium labelled antibody
A. 0.4mg CBL monoclonal antibodies are taken out and are placed in 30KD ultra-filtration centrifuge tubes, 8000rpm centrifugation 6min, discard filtrate.
B. mark buffer solution is added（0.1M PH9.0 carbonate buffer solutions）200ul, 8000rpm centrifuge 6min, discard filter
Liquid.Repeat this operation 4 ~ 5 times.
C. centrifuge tube filter membrane is inverted, 3000rpm centrifugation 6min, collects the antibody of concentration.
D. antibody is compared according to quality：Samarium chelate=4:1 ratio fully mixes, and is put into rotary incubator, room temperature reaction 16
~ 20 hours.
2. the purifying of samarium labelled antibody
Purify samarium mark CBL antibody with SepHadex TM G-50 sephadex columns, then add samarium labeling antibody preservative agent（8%
BSA+5%Proclin300）, make BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, 4 DEG C store for future use.
The above-mentioned samarium mark CBL antibody prepared is diluted by a certain percentage, dispensed to the hole of reagent strip the 4th（Label 2）In.
The excitation wavelength of described fluorescent material samarium is 340nm, launch wavelength 640nm.
（3）The preparation method of cleaning solution：
Prepare containing the .2% of 0 .8 ~ 1（v/v）Tween-20,0.05%（v/v）The 0.05M of Proclin300, pH 7.2 ~ 7.5
Tris-Hcl buffer solutions, the solution prepared is dispensed into washing lotion hole 6 and 7 with 900 μ L/ holes.
（4）Strengthen the preparation method of liquid：
Preparation contains 0.06%（w/v）Sodium acetate, 0.0001 ~ 0.0005%（w/v）β-NTA、0.0024% （w/v）TOPO 、
0.09%（v/v）Acetic acid, 0.1%（v/v）Absolute ethyl alcohol, 0.05%（v/v）Triton X-100 the aqueous solution.By what is prepared
Solution is dispensed to the hole of reagent strip the 12nd with 300 μ L/ holes（Strengthen fluid apertures）.
（5）Clenbuterol standard solution preparation method：
Totally 6 bottles of described Clenbuterol standard solution, concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7
μg/L、8.1μg/L。
The making and detection of the reagent strip of embodiment 3
Reagent strip in the present embodiment, semi-finished product are assembled by following process：Divide respectively in the 1st or 2,3 or 4,6,7,12
50 μ LBSA-CBL coatings magnetic bead, 200 μ L samariums labels, 900 μ L washing lotions, 900 μ L washing lotions, 300 μ L enhancing liquid are filled, then with envelope
Film machine seals, and is scribbled on described shrouding film and is available for the product information mark of full-automatic fluorimetric analysis instrument scanning recognition to include
Company standard curve, batch, date of manufacture, the term of validity.Finished product reagent strip, the Clenbuterol standard liquid dispensed and other match somebody with somebody
Part is assembled into kit.
Pattern detection：
Sample-adding
Sample to be tested or Clenbuterol standard items are put into the Load System of full-automatic instrument, reagent strip is inserted into reagent strip card
Groove, the product information of instrument automatic identification shrouding film.100 μ L samples to be tested or standard items are added in reagent strip 1 or 2, then
Add the μ L of fluorescent marker 100.
It is incubated
37 DEG C of concussions are incubated 15min after the completion of sample-adding.
Washing
After the completion of incubation, the automatic hole flushing of instrument 5 times, each μ L/ holes of washing lotion 200.
Add enhancing liquid
After the completion of washing, enhancing liquid 100 μ L/ holes, 37 DEG C of incubation 3min are added.
Detection
Reagent strip is pushed into darkroom, and instrument automatic detection simultaneously goes out result.
The specificity experiments of the kit of the present invention of embodiment 4
Using Clenbuterol as standard, if the cross reacting rate of Clenbuterol is 100%, for kit cross reaction Journal of Sex Research
Medicine be and Clenbuterol structure or intimate competition medicine：Salbutamol, Ractopamine, Cimaterol,
Clorprenaline, Tulobuterol, Bbu Tero, bricalin.Add above-mentioned 8 kinds of medicines (bag respectively in testing sample
Including clenbuterol) concentration is 6 μ g/L, by kit step operation, testing result is listed in table 1.As it can be seen from table 1 this reagent
Box has higher specificity to Clenbuterol, pair with Clenbuterol structure or it is intimate competition medicine without intersects instead
Should.
The kit specificity experiments of table 1
| Medicine name | Cross reacting rate |
| Clenbuterol | 100% |
| Salbutamol | 0 |
| Ractopamine | 0 |
| Cimaterol | 0 |
| Clorprenaline | 0 |
| Tulobuterol | 0 |
| Bbu Tero | 0 |
| Bricalin | 0 |
Sample loading system, bar code reading are included for the Full-automatic magnetic beads time-resolved fluorescence immunoassay instrument of test agent bar
System, sample adding system, incubation system, fluorescence light source detecting system and automatic software analysis and Control system.
Magnetic bead time-resolved fluoroimmunoassay detection kit of the present invention, reagent strip need to only be inserted when detecting sample
In full-automatic instrument, instrument is automatically performed sample-adding, incubation, Magneto separate, detection process, and whole process only needs 20min to read
Examining report.
The present invention detection urine sample or tissue extract, its sensitivity can reach 0.1ppb, the coefficient of variation within 5%,
It is higher than collaurum, fluorescence immune chromatography reagent sensitivity, more accurately.It is short compared to HPLC, GC/MS, ELISA detection time, valency
Lattice are cheap, can realize great amount of samples Site Detection.
Embodiment 5
In order to better illustrate beneficial effects of the present invention, be given below using detection reagent provided by the invention and GC/MS,
The Comparative result of HPLC, enzyme-linked immunosorbent assay, colloidal gold method, common fluorescent immunochromatographic method when detecting Clenbuterol is as follows
Table 2：
The different Determination of Clenbuterol performance comparisions of table 2
| Detection method | Required instrument | Detection time | Sensitivity | Precision |
| GC/MS | Gas chromatograph-mass spectrometer (GC-MS) | 3 ~ 4 hours | 0.5ppb | The coefficient of variation≤20% |
| HPLC | High performance liquid chromatograph | 3 ~ 4 hours | 0.5ppb | The coefficient of variation≤20% |
| Enzyme-linked immunosorbent assay | ELIASA | 2 hours | 0.3ppb | The coefficient of variation≤15% |
| Colloidal gold method | Naked eyes or colloidal gold chart reader | 10 ~ 20 minutes | 1ppb | The coefficient of variation≤20% |
| Common fluorescent immunochromatographic method | Fluorescence chart scanner | 15 ~ 20 minutes | 0.3ppb | The coefficient of variation≤15% |
| The method that kit of the present invention provides | Full-automatic time-resolved fluorescence immunoassay instrument | 20 minutes | 0.1ppb | The coefficient of variation≤5% |
Claims (10)
1. a kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit, it is characterised in that including BSA-CBL bags
By magnetic bead, fluorescent material mark CBL antibody, cleaning solution, enhancing liquid and Clenbuterol standard solution.
2. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
Described BSA-CBL coatings magnetic bead is the super-paramagnetic bead of 1 ~ 3 μm of diameter and the covalent coupling thing of albumen with modified with functional group,
The magnetic bead is carboxyl magnetic bead, amino magnetic bead, hydroxyl magnetic bead, tosyl magnetic bead, NHS magnetic beads, streptavidin magnetic bead, egg
One or more in white A magnetic beads, Protein G magnetic bead, anti-mouse IgG magnetic beads, hydrophilic magnetic bead, hydrophobic magnetic bead.
3. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
Described fluorescent material is group of the lanthanides huge legendary turtle and thing；The excitation wavelength of fluorescent material is 300 ~ 350nm, and launch wavelength is 500 ~ 650nm.
4. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 3 quantitatively detects Clenbuterol kit, it is characterised in that
Described fluorescent material is the chelate being made up of europium, samarium, terbium or dysprosium.
5. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
Described washing formula of liquid is：0 .8 ~ 1 .2% Tween-20,0.05% Proclin300, pH 7.2 ~ 7.5 0.05M
Tris-Hcl buffer solutions.
6. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
Described enhancing formula of liquid is：0.03% sodium acetate, 0.0001 ~ 0.0005% β-NTA, 0.0024% TOPO, 0.08% acetic acid,
The aqueous solution of 0.1% absolute ethyl alcohol, 0.05% Triton X-100.
7. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
Totally 6 bottles of described Clenbuterol standard solution, concentration are respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/
L、8.1μg/L。
8. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
The preparation method of described BSA-CBL coating magnetic beads：Carboxyl magnetic bead is put into centrifuge tube, centrifuge tube is placed on magnetic frame
In, the separation of magnetic bead and buffer solution is carried out using magnetic frame, with 0.05M pH6.0 2-（N- morpholines）Ethyl sulfonic acid washs magnetic bead
3 ~ 5 times；0.05M pH6.0 2- is added in the magnetic bead good to above-mentioned washing（N- morpholines）Ethyl sulfonic acid, add 30 μ L 10mg/
ML 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides solution and 30 μ L 50mg/ml n-hydroxysuccinimides,
Room temperature concussion reaction 0.5 ~ 1 hour；The magnetic bead activated is placed on magnetic frame, abandons supernatant, collects the magnetic bead activated；Add
BSA-CBL, room temperature persistently rotate incubation 30 ~ 240 minutes；Magnetic bead is placed on magnetic frame, abandons supernatant, collects immunomagnetic beads；Add
Enter the Tris-HCl buffer solutions containing 1%BSA 0.05M pH 8.0, magnetic bead is resuspended, closing is not coupled BSA-CBL activated carboxyl
Site, react 30 ~ 60min；Magnetic bead is placed on magnetic frame, abandons supernatant, 100 μ L immunomagnetic beadses is added and preserves liquid.
9. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 1 quantitatively detects Clenbuterol kit, it is characterised in that
It is the 0.05M containing 5%BSA, 5% sucrose, 0.1%Tween-20 and 0.1% polyvinylpyrrolidone that described immunomagnetic beads, which preserves liquid,
PH 8.0 Tris-HCl buffer solutions.
10. magnetic bead time-resolved fluoroimmunoassay as claimed in claim 4 quantitatively detects Clenbuterol kit, its feature exists
In the preparation method of described fluorescent material mark CBL antibody europium labeling antibodies is：CBL monoclonal antibodies are placed in 30KD ultrafiltration
Centrifuge tube, centrifugation, discards filtrate；Mark buffer solution is added, centrifugation, discards filtrate, repeats this operation 4 ~ 5 times；Centrifuge tube is filtered
Inversion membrane, centrifugation, the antibody of concentration is collected, mark buffer solution is added, stands, then centrifuge tube filter membrane is inverted, centrifuged, collect dense
The antibody of contracting；According to quality than Europium chelate DTPA-Eu=5:1 ratio fully mixes, and is put into rotary incubator, room temperature reaction；
Purify europium mark CBL antibody with SepHadex TM G-50 sephadex columns, then add europium labeling antibody preservative agent, make BSA and
Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, store for future use；
Or the preparation method of described fluorescent material mark CBL antibody samarium labeling antibodies is：CBL monoclonal antibodies are taken out to be placed in
30KD ultra-filtration centrifuge tubes, centrifugation, discard filtrate；Mark buffer solution is added, centrifugation, discards filtrate；Repeat this operation 4 ~ 5 times；Will
Centrifuge tube filter membrane inverts, centrifugation, collects the antibody of concentration；Compare antibody according to quality：Samarium chelate=4:1 ratio fully mixes,
Rotary incubator is put into, is reacted at room temperature 16 ~ 20 hours；Purify samarium mark CBL with SepHadex TM G-50 sephadex columns to resist
Body, samarium labeling antibody preservative agent is then added, makes BSA and Proclin300 final concentration of 0.2%, through 0.22 μm of membrane filtration, storage
It is standby.
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