CN102928592A - Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof - Google Patents
Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof Download PDFInfo
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- CN102928592A CN102928592A CN 201110227462 CN201110227462A CN102928592A CN 102928592 A CN102928592 A CN 102928592A CN 201110227462 CN201110227462 CN 201110227462 CN 201110227462 A CN201110227462 A CN 201110227462A CN 102928592 A CN102928592 A CN 102928592A
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Abstract
The present invention relates to a magnetic particle chemiluminescence kit for detecting trenbolone. The kit comprises a luminous marker, a fluorescein marker, a standard substance, a quality control substance and a separation reagent, wherein the luminous marker is isoluminol luminous marker labeled trenbolone hapten, the fluorescein marker is fluorescein labeled trenbolone monoclonal antibody or fluorescein derivative labeled trenbolone monoclonal antibody, and the separation reagent is sheep anti-FITC monoclonal antibody coated paramagnetic nanometer microbeads. The present invention further relates to a method for detecting trenbolone in foods of animal origin by using the kit, wherein the method provides characteristics of high sensitivity, high specificity and rapid detection for trenbolone detection.
Description
Technical field
The present invention relates to a kind of direct chemiluminescence detection kit and method of testing thereof.Particularly detect feed, aquatic products, urine, organize the magnetic particle of Trenbolone drug residue in the equal samples to compete direct chemiluminescence detection kit.
Technical background
Trenbolone chemistry trenbolone by name is artificial synthetic steroid class male sex hormone, also is the growth promoter of domestic animal, and the people is edible residual to have the meat products of this class material to bring harm to healthy.In addition, Trenbolone also becomes anti-depressant one of sports tournament and may originate.The food of this series products is used in European Union's import prohibition, also is that China customs forbids one of 31 kinds of veterinary drugs that detect.
At present, the detection method of Trenbolone has the methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrography (LC-MS) and enzyme linked immunosorbent assay (ELISA).
1, high performance liquid chromatography (HPLC) has the advantages that accuracy of detection is high, false positive rate is low.The major defect of HPLC method is that instrument is expensive, operate more loaded down with trivial details, length consuming time, testing cost is expensive, and testing staff's specialty is had relatively high expectations, Sample pretreatment is complicated.
2, liquid chromatography-mass spectrography (LC-MS), sample does not need derivatization treatment, can detect urine, blood, liver, hair and eyeball sample.The LC-MS/MS coupling can further improve signal to noise ratio (S/N ratio), so can be used for the affirmation means to positive findings.But, no matter LC-MS or LC-MS/MS, the instrument detection method is the same with GC-MS, and fails to solve instrument and involve great expense, complex operation step, Sample pretreatment is complicated, and operating personnel's Specialized Quality is required the problems such as high.
3, euzymelinked immunosorbent assay (ELISA) (ELISA) is that 20 century 70s occur, be used for the detection of micro substance, be applied to the earliest the clinical detection such as infectious disease, tumor markers, hormonal readiness, begin the nineties to apply in China food safety detection field, relying at present the kit of elisa technique, the leading products that test strips has become food security fast detecting field, also is simultaneously the major product type that my company researches and develops and sells.The enzyme linked immunosorbent detection method is based on the specific reaction of Ag-Ab, detection sensitivity is higher, specificity is better, technical operation is simple and easy, easily grasp, really solved the qualitative and quantitative analysis work of a large amount of former insoluble many micro-small-molecule substances such as microbiotic, hormone, residues of pesticides etc., positive facilitation has been played in the development of food security Fast Detection Technique.But there are many defectives that self can't overcome in the ELISA method, and is mainly manifested in:
1) heterogeneous reaction: be separated free thing and bond in the testing process, need multistep to wash the plate process, time and effort consuming is difficult to improve the automaticity of operation.
2) enzymic catalytic reaction: by substrate for enzymatic activity colour developing, assaying reaction liquid absorbance.There are considerable influence in time and the temperature of reaction to enzyme activity, and reagent stability is poor.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of magnetic granule chemiluminescence kit that detects Trenbolone, not only possesses higher sensitivity when adopting this kit to carry out the residual detection of Trenbolone, specificity, and have higher reaction velocity.
The method of testing that provides a kind of Trenbolone residual is provided another object of the present invention, and the method is simple to operate, and is highly sensitive, and specificity is good.
Realize above-mentioned purpose, the invention provides a kind of Trenbolone detection kit, its main agents that comprises has: luminous marker, fluorescein-labelled thing, standard items, quality-control product, separation agent.
Described separation agent is the paramagnetism nano microsphere that is coated with goat-anti FITC antibody.
Described paramagnetism nano microsphere, surface contain-OH ,-COOH or-NH
2The microballon of reactive group is used for coated goat-anti FITC monoclonal antibody, and its inner core is Fe
3O
4Or γ-Fe
2O
3, so that microballon has paramagnetism.
Described luminous marker is the Trenbolone haptens of different Derivative of Luminol mark.Described different Derivative of Luminol is ABEI, AHEI or ABEN.
Described kit also comprises standard items, quality-control product and concentrated washing lotion.
Described fluorescein-labelled thing is FITC mark Trenbolone monoclonal antibody.
The present invention also provides a kind of method of utilizing kit to detect Trenbolone, comprises the following steps:
1) draw respectively 20 μ l-100 μ l standard items or samples, then add 20 μ l-100 μ l luminous markers, add again the fluorescein-labelled thing of 20 μ l-100 μ l, behind the abundant mixing, 37 ℃ of incubation 15min;
2) add the paramagnetism nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min;
3) separate 5min with the magnetic separator frame, abandon behind the supernatant with cleaning fluid 300-500 μ l flushing compound precipitation;
4) above-mentioned cleaning step repeats 2-4 time;
5) 4) compound of gained separator well is directly put into the measurement camera bellows, add and excite substrate 1 and excite substrate 2, postpone to detect the relative light intensity (RLU) that sends behind the 3-5s, content in the sample and RLU proportion relation can be gathered the concentration that calibration curve method calculates Trenbolone by RLU.
The principal ingredient of described luminous substrate is NaOH and H
2O
2
The used analyser of analysis test method is among the present invention: comprise power circuit, automatic injection pump 1 and 2, measuring chamber, illuminated chamber, photomultiplier counter and output system, also dispose simultaneously the Windows control software of computing machine and Chinese interface, can carry out that data typing, result gather, quality control, the result stores and the function such as result queries, can finish the programming of multiple analytical model, quantitative or qualitative reporting the result, automatic generation and storage, update functions, automatically revise typical curve at 2, the system that adopts is the CI-2008 system.
Separation agent of the present invention is coated goat-anti FITC monoclonal antibody, and the content of its surface group is 0.1-0.3eqm/g, and it is stored in pH7.2, contains the 3-5% ovalbumin, 0.1-0.3%Tween-20,0.4%NaN
3The PBS damping fluid in.Described FITC is fluorescein isothiocynate.Described percentage composition is the quality percentage composition.
Described luminous marker is the Trenbolone haptens of different Derivative of Luminol ABEI, AHEI or ABEN mark, and it is stored in and contains pH7.2,0.3%Tween-20,0.1%NaN
3The PBS damping fluid in.Described percentage composition is the quality percentage composition.
The Trenbolone monoclonal antibody that described fluorescein-labelled thing is the FITC mark, it is stored in pH7.2, contains the 4-6% ovalbumin, 0.2-0.4%NaN
3The PBS damping fluid.Described percentage composition is the quality percentage composition.
Trenbolone standard solution (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml), the standard items dilution is pH7.4,0.03%NaN
3, the 0.1mol/LPBS damping fluid.Described percentage composition is the quality percentage composition.
Trenbolone quality-control product solution concentration is respectively 0.02ng/ml, 0.8ng/ml, quality-control product dilution pH7.4,0.03%NaN
3, the 0.1mol/LPBS damping fluid.Described percentage composition is the quality percentage composition.
Described concentrated washing lotion is PH7.2,0.2-0.4%Tween-20,0.2-0.4%NaN
3, the 0.1-0.2mol/LPBS damping fluid.Described percentage composition is the quality percentage composition.
Beneficial effect of the present invention is as follows:
1) but kit specific detection Trenbolone of the present invention, to other drug without intersection.
2) sensitivity of kit of the present invention is higher, can reach 0.01ng/ml to the detection sensitivity of Trenbolone.
3) detection of kit of the present invention is quick, is lower than 20min detection time.
Embodiment
The preparation of the concrete component of embodiment one kit
1) Trenbolone is haptenic synthetic
Trenbolone is synthesized the Trenbolone haptens with the succinic anhydride method.
2) preparation of artificial antigen
Adopt carbodiimide method to carry out coupling Trenbolone haptens and bovine serum albumin and all arrive immunogene.
3) preparation of monoclonal antibody
Animal immune: with immunogene the Balb/c mouse is carried out immunity, immunizing dose is 100 μ g/, makes it produce antiserum.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, merge in 9: 1 ratios and SP2/0 myeloma cell, obtain the hybridoma cell strain of monoclonal antibody.
Cell cryopreservation and recovery: hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
The preparation and purification of monoclonal antibody: the Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
7Individual/as only, to gather ascites after 7 days.Carry out purifying, obtain monoclonal antibody with sad-saturated ammonium sulfate method.
4) preparation of luminous marker
Get 4.5mmol/L ABEI, be dissolved in the 4ml distilled water, the 5.0mmol/L N-hydroxy-succinamide is dissolved in the 0.5ml DMF, room temperature reaction 3-4h behind the two abundant mixing.Get the Trenbolone haptens 15mg of above-mentioned preparation, to 1.5ml, then add the ABEI solution of above-mentioned activation with the pH7.4PBS adjusted volume, fully room temperature reaction spends the night behind the mixing, crosses G-25 gel column purifying.
5) fluorescent marker preparation
Use 0.025mol/L, the carbonate buffer solution of pH9.0 is diluted to 1% mass percent concentration with the Trenbolone monoclonal antibody; Total protein according to wanting mark adds 0.01mg FITC by every milligram of immunoglobulin (Ig), accurately takes by weighing required FITC powder with analytical balance.FITC is made into the solution of 0.1mg/ml with same damping fluid, by 3-5 times of above-mentioned antibody-solutions volume, sneaks into the FITC dilution; Electromagnetic agitation, mark 30-48h under 4～℃ lucifuge condition; With label solution 3000r/min, the centrifugal 20min of room temperature removes wherein a small amount of sediment, and in the bag filter of packing into, the PBS damping fluid of pH7.4 dialysis is 2-3 days again, during change at least dislysate 3 times; Get the label of dialysed overnight, by SephadexG-25 or G-50 post, the separated free fluorescein is collected the fluorescent marker of mark and is identified that packing is stored in 4 ℃ of refrigerators.
6) separation agent preparation
A) magnetic bead activation
The magnetic bead of surface-COOH group (be purchased from DYNAL, particle diameter is 2.8 μ m), its content is 0.15eq/g; Get 100 μ l magnetic beads, with 100 μ l 25mmol/L, pH5.0,0.05%Tween-20MES solution washing twice, magnetic removes supernatant after separating; Before the use, configure respectively EDC, the NHS solution of 50mmol/L with the 25mmol/L MES solution of 4 ℃ of storages; Add respectively the new EDC that configures of 50 μ l and NHS solution in the centrifuge tube that magnetic bead is housed, vortex mixing, room temperature activation 30min; Centrifuge tube places on the magnetic separator frame magnetic to separate 4min, removes supernatant, adds 100 μ l, and 25mmol/L, pH5.0, MES clean and get final product to get the magnetic bead of surperficial activated carboxylic after 2-3 time.
B) magnetic bead coupling goat-anti FITC monoclonal antibody
50-100 μ g goat-anti FITC monoclonal antibody is dissolved into 60 μ l, and 25mmol/L among the pH5.0MES, regulates cumulative volume to 100 μ l with described MES solution, soft mixing magnetic bead and antibody; At least coupling 30min or 4 ℃ of coupling 2h under the room temperature condition can utilize the vortex instrument to make magnetic bead keep the mixing state during this period; Centrifuge tube places magnetic separation 3-5min on the magnetic separator frame, removes supernatant; For cancellation unreacted-COOH, can add 100 μ l, pH7.4, TRIS reaction 15min or 100 μ l, pH8.0 contains the PBS sealing magnetic bead of 50mmol/L monoethanolamine; With 100 μ l, 0.1-0.3%BSA, the PBS of 0.1%Tween-20 or TRIS clean the good magnetic bead of sealing 3-5 time; At last magnetic bead is redissolved in containing 0.1-0.5%BSA, 0.01-0.1%Tween-20,0.02%NaN
3PBS or the TRIS damping fluid in, 2-8 ℃ of preservation.
The establishment of embodiment two kits
Set up the magnetic granule chemiluminescence kit that detects Trenbolone, make it contain following component:
The fluorescent marker of the Trenbolone monoclonal antibody of FITC mark
The haptenic luminous marker of the Trenbolone of ABEI mark
The separation agent of the paramagnetism nano microsphere of pan coating goat-anti FITC monoclonal antibody
Trenbolone standard solution (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml), the standard items dilution is pH7.4,0.03%NaN
3, the 0.1mol/LPBS damping fluid.Described percentage composition is the quality percentage composition.
Trenbolone quality-control product solution concentration is respectively 0.02ng/ml, 0.8ng/ml, quality-control product dilution pH7.4,0.03%NaN
3, the 0.1mol/LPBS damping fluid.Described percentage composition is the quality percentage composition.
Concentrated washing lotion is PH7.2,0.2-0.4%Tween-20,0.2-0.4%NaN
3, 0.1-0.2mol/L PBS damping fluid.Described percentage composition is the quality percentage composition.
The detection of Trenbolone in embodiment three actual samples
1, Sample pretreatment
(1) feed, tissue (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method
With homogenizer homogeneous structure, feed sample; Take by weighing the equal pledge of 2.0 ± 0.05g and add 10ml acetonitrile-0.1M sodium hydroxide solution to the 50ml polystyrene centrifuge tube, with the oscillator 10min that vibrates, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); Pipette the 0.5ml supernatant to 50ml polystyrene centrifuge tube, add 0.5ml 0.1M sodium hydroxide solution, add the 5ml methenyl choloride after the vibration, with vortex instrument whirling motion 2min, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃).(if emulsion appears in sample to be needed at 70 ℃ of water-bath 2-4min, again centrifugal until lower floor limpid till); Remove the upper strata, get the limpid organic phase of 1ml lower floor to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; With the dry residue of 1ml redissolution liquid working fluid dissolving; Getting 50 μ l waters is used for analyzing sample extension rate: 50.
(2) urine pre-treating method
Get the 2ml urine in centrifuge tube, more than 3000g, the centrifugal 5min of room temperature (20-25 ℃) is until urine specimen is limpid with urine; Pipette the limpid urine specimen of 1ml to 50ml polystyrene centrifuge tube, add 10 μ lGlucuronidase/Arylsulfatase (glucuronidase/aryl sulfatase), at 37 ℃ of hydrolysis 2h, add the 5ml methenyl choloride, with the oscillator 5min that vibrates, more than the 3000g, the centrifugal 10min of room temperature (20-25 ℃); The limpid organic phase of 1ml lower floor is got to the glass test tube of 10ml drying in the removal upper strata, flows down in 50～60 ℃ of water-bath nitrogen to dry up; With the dry residue of 1ml redissolution working fluid dissolving; Getting 50 μ l waters is used for analyzing sample extension rate: 5.
2, detect and interpretation of result with kit
Draw respectively 20 μ l-100 μ l standard items or samples, then add 20 μ l-100 μ l luminous markers, add again the fluorescein-labelled thing of 20 μ l-100 μ l, behind the abundant mixing, 37 ℃ of incubation 15min; Add the paramagnetism nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min; Separate 5min with the magnetic separator frame, abandon behind the supernatant with cleaning fluid 300-500 μ l flushing compound precipitation; The compound of gained separator well is directly put into the measurement camera bellows, add and excite substrate 1 and excite substrate 2, postpone to detect the relative light intensity (RLU) that sends behind the 3-5s, the content of Trenbolone and RLU proportion relation in the sample can be by the concentration of RLU combined standard curve method calculating Trenbolone.
Exciting substrate 1 is NaOH, and exciting substrate 2 is H
2O
2Before substrate loads, clean the substrate pump 10-20 time with distilled water, behind the interior remaining water mark of emptying pipe, more corresponding substrate is directly put into instrument, pump line is inserted in the substrate bottle; With substrate flushing pipe 5 times, and measure the RLU value of substrate, under normal circumstances, the RLU value of substrate should be above 1200.If surpass 1200, need again with distilled water pipeline and substrate pump to be carried out more times cleaning, until blank value is down in the zone of reasonableness.Do not do experiment if surpass three days, need unloading substrate bottle, and cover lid, with vaporization prevention.Use subsequently the distilled water pipe blow-through, and emptying, in order to avoid strong base solution corrosion substrate pump.
The present invention adopts 6 Trenbolone standard items (0ng/ml, 0.01ng/ml, 0.03ng/ml, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml) to carry out plotting curves.Instrument detects according to described method and obtains a RLU value and the concentration dependent calibration curve of Trenbolone, and after this in the measurement, the Trenbolone concentration in each sample is compared with typical curve and drawn Trenbolone content in the sample.
The mensuration of embodiment four kit quality
1, the sensitivity of kit
Kit sensitivity is defined as: measure 20 times the zero standard product, the mean value of mensuration adds 3 times of standard deviations.The sensitivity of this kit is 0.01ng/ml.
2, the accuracy of sample and precision
Accuracy refers to the matching degree between measured value and true value, and the kit accuracy recovery commonly used represents.Precision claims again repeatability, and the coefficient of variation commonly used represents.
Sample extraction method according to embodiment three, Trenbolone with 1ng/g, two concentration of 2ng/g is added chicken, Trenbolone with 0.1ng/ml, two concentration of 0.2ng/ml is added recovery to urine specimen, every kind of each 4 of each concentration of sample are parallel, measure with three batches of kits, calculate average recovery rate and the precision of sample.Experimental result sees the following form.
Table 1 accuracy and precision are measured ng/g (ml)
As seen from the table, the average recovery rate scope that two concentration of Trenbolone are all added in chicken, the urine sample between 84.5-96.8%, in batch, batch between all the coefficient of variation less than 15%.
3, specificity
Cross reacting rate refers to the ability of the antigenic determinant generation combination that antibody is different from structure.
Table 2 kit cross reacting rate
| Medicine name | Cross reacting rate |
| Trenbolone | 100％ |
| The dehydrogenation epitestosterone | Less than 1% |
| Testosterone | Less than 1% |
| Metandienone | Less than 0.1% |
| The 19-nortestosterone | 4.5％ |
4, correlativity
X＝CI-2008，Y＝RIA
Y＝1.25X-2.79
R＝0.9813
X is CI-2008 system measurement result, and Y is the ria-determination result.
Claims (8)
1. magnetic granule chemiluminescence kit that detects Trenbolone, the reagent that comprises has: luminous marker, fluorescein-labelled thing, standard items, quality-control product, separation agent.
2. kit according to claim 1, it is characterized in that: described separation agent is the paramagnetism nano microsphere that is coated with goat-anti FITC monoclonal antibody.
3. kit according to claim 2 is characterized in that: described paramagnetism nano microsphere is that the inside is coated with Fe
3O
4Or γ-Fe
2O
3, the surface contains-OH ,-COOH or-NH
2The microballon of reactive group.
4. each described kit according to claim 1-3, it is characterized in that: described luminous marker is the Trenbolone haptens of different Derivative of Luminol mark.
5. kit according to claim 4, it is characterized in that: described testosterone coupled isoluminol thing is ABEI, AHEI or ABEN.
6. each described kit according to claim 1-3, it is characterized in that: described kit also comprises titer, quality-control product and concentrated washing lotion.
7. one of according to claim 1-3 described kit is characterized in that: the Trenbolone monoclonal antibody that described fluorescein-labelled thing is the FITC mark.
8. a method of utilizing each described kit of claim 1-7 to detect Trenbolone comprises the following steps:
1) draw respectively 20 μ l-100 μ l standard items or samples, then add 20 μ l-100 μ l luminous markers, add again the fluorescein-labelled thing of 20 μ l-100 μ l, behind the abundant mixing, 37 ℃ of incubation 15min;
2) add the paramagnetism nano microsphere 80-150 μ l be coated with goat-anti FITC monoclonal antibody, behind the mixing at 37 ℃ of incubation 5min;
3) separate 5min with the magnetic separator frame, abandon behind the supernatant with cleaning fluid 300-500 μ l flushing compound precipitation;
4) above-mentioned cleaning step repeats 2-4 time;
5) 4) compound of gained separator well is directly put into the measurement camera bellows, add and excite substrate 1 and excite substrate 2, postpone to detect the relative light intensity (RLU) that sends behind the 3-5s, the content of Trenbolone and RLU proportion relation in the sample can be gathered the concentration that calibration curve method calculates Trenbolone by RLU.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110227462 CN102928592A (en) | 2011-08-09 | 2011-08-09 | Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110227462 CN102928592A (en) | 2011-08-09 | 2011-08-09 | Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof |
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| CN102928592A true CN102928592A (en) | 2013-02-13 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116031A (en) * | 2013-02-20 | 2013-05-22 | 河南科技学院 | Quick trenbolone residue detection test paper card and preparation method thereof |
| CN107643407A (en) * | 2017-09-16 | 2018-01-30 | 北京勤邦生物技术有限公司 | A kind of magnetic immunochemiluminescence detection kit of Trenbolone and its application |
-
2011
- 2011-08-09 CN CN 201110227462 patent/CN102928592A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116031A (en) * | 2013-02-20 | 2013-05-22 | 河南科技学院 | Quick trenbolone residue detection test paper card and preparation method thereof |
| CN107643407A (en) * | 2017-09-16 | 2018-01-30 | 北京勤邦生物技术有限公司 | A kind of magnetic immunochemiluminescence detection kit of Trenbolone and its application |
| CN107643407B (en) * | 2017-09-16 | 2020-08-28 | 北京勤邦生物技术有限公司 | Trenbolone magnetic immunochemiluminescence detection kit and application thereof |
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Application publication date: 20130213 |