CN1793927A - Reagent box for detecting clenbuterol hydrochloride and its detection method - Google Patents
Reagent box for detecting clenbuterol hydrochloride and its detection method Download PDFInfo
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- CN1793927A CN1793927A CN 200510097304 CN200510097304A CN1793927A CN 1793927 A CN1793927 A CN 1793927A CN 200510097304 CN200510097304 CN 200510097304 CN 200510097304 A CN200510097304 A CN 200510097304A CN 1793927 A CN1793927 A CN 1793927A
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Abstract
A method for detecting CBL includes competing CBL antibody by tree CBL with CBL - OVA on micro hole board, washing off unconnected CBL antibody, adding EU3+ - sheep resisting rabbit antibody and washing off unconnected EU3+ - sheep resisting rabbit antibody, adding intensified liquid and using time - identification luminoscope to determine its fluorescence intensity, presenting fluorescence intensity to CBL concentration in sample to be inverse ratio, comparing with standard curve to obtain CBL content in sample. The reagent kit for realizing said method is also disclosed.
Description
Technical field
A kind of kit and detection method thereof that detects clenobuterol hydrochloride belongs to time resolved fluoro-immunoassay (TRFIA) technical field, is used for the detection to animal food, blood and urine clenobuterol hydrochloride (being called for short CBL) content.
Background technology
Clenobuterol hydrochloride (CBL) is a kind of beta-2-adrenoceptor activator, clinically as the bronchus spasmolysis medicine, bronchial smooth muscle β-2 acceptor is had excitation.Studies show that clenobuterol hydrochloride can strengthen the steatolysis and the protein catabolism that slows down, so if heavy dose of use in herding is produced can obviously be improved food conversion ratio and lean meat percentage, clenbuterol hydrochloride therefore is otherwise known as.If but using dosage is excessive, then can produce sizable toxic action to liver, the kidney and other organs of animal and human's (indirectly), dizziness, palpitaition, expiratory dyspnea, muscular tremor, toxicity symptoms such as headache appear palpitating quickly.Clenobuterol hydrochloride also can enter to produce in the fetus body by placental barrier to be accumulated, thereby filial generation is produced serious harm.
European Union forbade in 1996 using this medicine (EC Directive 96/22/EC) in herding is produced, and identical regulation was also made by China Ministry of Agriculture in 1997.However, because ordering about of economic interests uses this phenomenon of clenobuterol hydrochloride to still have generation at present, extensive poisoning all takes place every year in herding is produced.The pollution of clenobuterol hydrochloride has caused the fear of people to foodsafety.Current, causing one of reason that clenbuterol hydrochloride remains incessant after repeated prohibition is exactly also imperfection of the method that detects clenobuterol hydrochloride both at home and abroad, means, and the problem of existence mainly contains: sensitivity is not high, and accuracy is not enough; Poor specificity, technical sophistication can not spread to basic unit.
The detection method of clenobuterol hydrochloride mainly contains at present: chromatographic technique and immuno analytical method.Chromatographic technique (HPLC, GC-MS, LC-MS) is because time-consuming, and cost is many, is subjected to certain limitation in actual applications, usually as deterministic quantitative test method; Immuno analytical method (ELISA) is the scene sampling rapid screening method of inspection of using always, at present, China imports and exports inspection and quarantine, the German R-Biopharm of the many employings in veterinary hygiene department and slaughterhouse, the clenobuterol hydrochloride ELISA detection kit that companies such as Britain Randox produce, but, limited its usable range to a certain extent because the import reagent box costs an arm and a leg.Problems such as homemade ELISA kit ubiquity insufficient sensitivity, false positive rate height, instability.
Time resolved fluoro-immunoassay method (TRFIA) is the new immunoassay that last century, early eighties grew up.Its principle of time resolved fluoro-immunoassay method is to utilize the sequestrant with bifunctional group structure, one end and lanthanide series combination, and the free amino group on the other end and the antibody molecule connects, and makes EU
3+Antigen in the labelled antibody, it and testing sample is combined into immune complex.Ideally, the fluorescence intensity of measuring lanthanide series in the compound just can be determined the amount of antigen in the sample, but the fluorescence intensity of in fact this compound quite a little less than, have only and add a kind of enhancing solution (Enhancement solution) again, lanthanide series is disintegrated down from compound, and with strengthen β-naphthoyltrifluoroacetone contained in the liquid (β-NTA) form microcapsules again, the very strong fluorescence of emission under the exciting of light such as ultraviolet, up to a million times of enhancing effects.Differentiate luminoscope with the time and measure its fluorescence intensity cps, can determine the amount of antigen in the sample.
Summary of the invention
The object of the present invention is to provide kit and the detection method thereof of a kind of CBL of detection, be used for detection animal food, blood and urine CBL content.
Technical scheme of the present invention: the kit of this detection CBL be by 1.96 or 48 holes bags by plate, 2. damping fluid, 3. clenobuterol hydrochloride standard, the antibody dried frozen aquatic products of 4 clenobuterol hydrochlorides, the 5. goat anti-rabbit antibody of europium mark, 6. cleansing solution 7. strengthens liquid and forms.
Bag is by plate (1): bag is used 50mmol/L pH9.6Na by solid phase antigen
2CO
3-NaHCO
3Damping fluid clenobuterol hydrochloride-ovalbumin is diluted to 1mg/L as coating buffer, 96 or 48 each hole of hole microwell plate add 100 μ l, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the damping fluid sealing of Tris-HCl that 200 μ l contain the 50mmol/LpH7.2 of mass concentration 0.2% gelatin, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing;
Damping fluid (2): 8mmol/L NaCl, mass concentration 0.1% gelatin, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and contain mass concentration 0.1%NaN
350mmol/L Tris-HCl pH7.8;
Clenobuterol hydrochloride standard (3): totally 6 bottles, clenobuterol hydrochloride concentration is respectively: 0ng/ml, and 0.05ng/ml, 0.25ng/ml, 1ng/ml, 5ng/ml, 25ng/ml, dilution are the PBS of pH7.4 10mmol/L;
The antibody dried frozen aquatic products (4) of clenobuterol hydrochloride: with Freund's complete adjuvant or incomplete Freund 1.2ml mixing 2mg clenobuterol hydrochloride-bovine serum albumin(BSA), mixed 2 hours with homogenizer, make Water-In-Oil antigen emulsifying agent, get the Water-In-Oil antigen emulsifying agent that 600 μ l prepare, multidigit point ground carries out hypodermic injection on one's body new zealand white rabbit, after immunity 3～4 times, serum identifies, qualified back dilution, packing, freeze-drying are standby;
The goat anti-rabbit antibody of europium mark (5): get the 5g/L goat anti-rabbit antibody 1-2ml that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/LNa that contains 0.155mol/L NaCl
2CO
3-NaHCO
3The pH8.5-9.0 damping fluid is collected protein peak, and is quantitative through the uv absorption analysis, and to 2g/L, the goat anti-rabbit antibody of getting after 500-1000 μ l dilutes adds the Eu that contains 0.2-0.4mg with above-mentioned eluent dilution goat anti-rabbit antibody
3+-N
2In the bottle of-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, 28-30 ℃ of magnetic agitation reacted 16-20 hour, reactant liquor is collected protein peak through the 1 * 40cmSephadex-G50 column chromatography with 80mmol/L Tris-HCl pH7.8 damping fluid balance, and dilution, packing are standby;
Cleansing solution (6): 14.5mmol/L NaCl, 0.2ml/L Tween-80 and contain mass concentration 0.2%NaN
350mmol/L Tris-HCl pH7.8;
Strengthen liquid (7): 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1ml triton x-100.
The present invention mainly adopts time resolved fluoro-immunoassay method (TRFIA) to detect CBL.Adopt the technology of TRFIA to mainly contain two aspects: the preparation of specific polyclonal antibody, utilize the antigen immune rabbit, obtain to contain the serum of antibody, through biochemical purification separating immune globulin; The second, EU
3+The preparation of labelled antibody.
Assay method is: the basis of mensuration is the labelled immune reaction.Be coated with the microwell plate of CBL-OVA, the sample that adds the CBL standard or handled well in micropore separately, add CBL antibody again, oscillating reactions, free CBL and the CBL-OVA on the microwell plate compete CBL antibody, the cleansing solution washing, the CBL antibody that does not have to connect is removed in washing step.Add EU
3+-goat anti-rabbit antibody carries out the labelled immune reaction, and with the cleansing solution washing, the reaction back does not have the EU of connection again
3+-goat anti-rabbit antibody is removed in washing step.After adding the vibration of enhancing liquid, the very strong fluorescence of emission under the exciting of ultraviolet light is differentiated luminoscope with the time and is measured its fluorescence intensity cps, and the CBL concentration in fluorescence intensity and the sample is inversely proportional to, the clenobuterol hydrochloride content in the reference standard curve calculation sample.
It is operating as: get be coated with clenobuterol hydrochloride-ovalbumin the micropore bag by plate, the sample that adds the clenobuterol hydrochloride standard of 50 μ l or handled well is in micropore separately, add with damping fluid (2) and make thinning agent, 50 μ l antibody of clenbuteral hydrochloride after diluting 20 times at 1: 20,25-37 ℃ vibrated 0.5-1 hour, cleansing solution is given a baby a bath on the third day after its birth time, adds with 100 μ l EU after 20 times of damping fluid (2) dilutions in 1: 20
3+-goat anti-rabbit antibody, 25-37 ℃ vibrated 0.5-1 hour, washed six times with cleansing solution, added the vibration of 200 μ l enhancing liquid and measured fluorescence intensity cps, the content of clenobuterol hydrochloride in the reference standard curve calculation sample after 5 minutes.
Sample preparation, animal tissue's sample preparation: with reference to GB/T5009.192-2003; Blood sample is handled: gets the PBS that 0.5ml blood adds 0.5mlpH7.4 10mmol/L and dilutes, and standby; Urine sample is handled: limpid pig urine sample is diluted more than 10 times with the PBS of pH7.4 10mmol/L, and is standby, if the urine sample muddiness must be filtered or be centrifugal, gets filtrate or supernatant and dilutes back standby.
Beneficial effect of the present invention: this kit is simple in structure, and is easy to use, cheap, highly sensitive, and minimal detectable concentration is less than 0.05ng/ml, and corresponding lowest detection amount is less than 25ng/kg.
Description of drawings
Fig. 1: the kit synoptic diagram that detects clenobuterol hydrochloride.1.96 or 48 holes bags is by plate, 2. damping fluid, and 3. clenobuterol hydrochloride standard, the 4. antibody dried frozen aquatic products of clenobuterol hydrochloride, the 5. goat anti-rabbit antibody of europium mark, 6. cleansing solution 7. strengthens liquid.
Fig. 2: CBL-TRFIA canonical plotting.
Embodiment
Embodiment 1 preparation kit and detection liver sample
CBL is typical haptens, thus in immune response, have reactionogenicity, but need and immunogenicity is just arranged after macromolecular substances combines, the carboxyl among the CBL is a reactive group, can combine with the amino of protein.Therefore available diazo coupling method makes CBL combine with high molecular weight protein bovine serum albumin(BSA) (BSA), carries out animal immune with the CBL-BSA that synthesizes as the immunizing antigen of synthetic.
The preparation of CBL-BSA antigen: 3-6mgCBL is dissolved among the HCl of 100mmol/L precooling, slowly drips the 1mol/L sodium nitrite solution, checks when test paper becomes darkviolet with the KI starch paper to stop, and obtains the diazotising derivant.Taking by weighing 10-20mgBSA is dissolved in the carbonate buffer solution of pH9.650mmol/L.Diazotizing CBL is slowly splashed in the BSA solution, regulate pH with 1mol/LNaOH and maintain 9.0～9.5, after dripping end, continue reaction 4 hours, phosphate buffer (PBS) with pH7.4 10mmol/L in 4 ℃ of refrigerators was dialysed two days, obtain the CBL-BSA bond and carry out the UV scanning detection, qualified back packing is standby.
The preparation of polyclone antibody of clenbuteral hydrochloride:
1. it is big to choose for 4 weeks, the healthy new zealand white rabbit of the about 1.5Kg of body weight.CBL is a kind of haptens, and CBL is connected as antigen with BSA.
The preparation of 2 Water-In-Oil antigen emulsifying agents:, made Water-In-Oil antigen emulsifying agent in 2 hours with the homogenizer mixing with Freund's complete adjuvant or incomplete Freund 1.2ml mixing 2mgCBL-BSA.The emulsifying agent that makes is splashed in the beaker that fills cold water, and an oil droplet state can intactly rest on the water surface, and indiffusion shows it is stable.
3. immune rabbit and blood drawing: the hair with the rabbit back carefully cuts off earlier, get the Water-In-Oil antigen emulsifying agent that 600 μ l prepare then, multidigit point ground carries out hypodermic injection on one's body new zealand white rabbit, antigen can slowly be spread, every 1～2 all immunity once, need six times altogether, after immunity 3～4 times, from the about 1ml of auricular vein blood drawing of rabbit, behind the centrifugal 10min, getting serum can identify.The dilution of qualified back, packing, freeze-drying are standby.
Eu
3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 1-2ml that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/L Na that contains 0.155mol/L NaCl
2CO
3-NaHCO
3The pH8.5 damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.The goat anti-rabbit antibody of getting after 500-1000 μ l dilutes adds the Eu that contains 0.2-0.4mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through Sepharose CL-6B post (1 * 40cm) chromatography, A with 80mmol/L Tris-HCl pH7.8 damping fluid balance
280Protein peak is collected in monitoring, and the dilution packing is standby.
Bag is prepared by the plate solid phase antigen:
Use 50mmol/L Na
2CO
3-NaHCO
3The pH9.6 damping fluid is diluted to 1mg/L as coating buffer with CBL-BSA, and each hole of microwell plate, 96 (or 48) hole adds 100 μ l, and 4 ℃ of placements are spent the night.Discard coating buffer, wash three times, add the damping fluid sealing of Tris-HCl that 200 μ l contain the 50mmol/LpH7.2 of mass concentration 0.2% gelatin, 4 ℃ of placements are spent the night.Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent:
(1) clenobuterol hydrochloride standard, totally 6 bottles, concentration is respectively: 0ng/ml, 0.05ng/ml, 0.25ng/ml, 1ng/ml, 5ng/ml, 25ng/ml, dilution are the PBS of pH7.4 10mmol/L.
(2) damping fluid: 8mmol/L NaCl, mass concentration 0.1% gelatin, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and contain mass concentration 0.1%NaN
350mmol/L Tris-HCl pH7.8.
(3) cleansing solution: 14.5mmol/L NaCl, 0.2ml/L Tween-80 and contain mass concentration 0.2%NaN
350mmol/L Tris-HCl pH7.8.
(4) strengthen the preparation of liquid: 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-naphthoyltrifluoroacetones (β-NTA), 50 μ mol trioctyl-phosphine oxide (TOPO), 1ml triton x-100 (TritonX-100).
The reagent that kit provides
Reagent in each box enough carries out 96 measurements, and the material in the box is as follows:
(1) .1 * 96 orifice plates (8 * 12 hole can be split as single hole) are coated with CBL-OVA.
(2) .6 * CBL titer, the 1.0ml/ bottle, concentration of standard solution is: 0,0.05,0.25,1,5,25ng/ml.
(3) .1 * CBL antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
(4) .1 * EU
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
(5) .1 * enhancing liquid: 15ml.
(6) .1 * cleansing solution: 30ml, the time spent is with distilled water dilution in 1: 25.
(7) .1 * damping fluid: 30ml,
The reagent that the laboratory should be provided for oneself
Perchloric acid
Isopropyl alcohol
Ethyl acetate
Perchloric acid solution (0.1mol/L)
Sodium hydroxide solution (1mol/L)
The PBS damping fluid (pH7.4,0.01mol/L)
Isopropyl alcohol+ethyl acetate (volume ratio 40+60)
Distilled water or deionized water.
Points for attention before measuring
1. before using all reagent are gone up to room temperature (18-30 ℃).
2. immediately all reagent are put back to 2-8 ℃ after using.
3. if the hyperchannel pipettor is used in the big suggestion of sample size.
4. hatch in the process at all constant temperature, avoid irradiate light, use the cap covers micropore.
5. taking-up needs microwell plate and the framework with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2-8 ℃.
Concrete detection step is as follows:
Earlier sample is handled: animal tissue's sample preparation, with reference to GB/T5009.192-2003.Take by weighing liver sample 10g (being accurate to 0.01g), with the homogenate of 20mL0.1mol/L perchloric acid solution, place the grournd glass centrifuge tube, place the ultrasonic 20min of ultrasonic cleaner then, taking-up places 80 ℃ of water-baths to heat 30min, takes out cooling back centrifugal (4500r/min) 15min.Inclining supernatant, and precipitation is with the washing of 5mL0.1mol/L perchloric acid solution, and is centrifugal again, and twice supernatant is merged.With 1mol/L sodium hydroxide solution adjust pH to 9.5 ± 0.1, if there is precipitation to produce, centrifugal again (4500r/min) 10min, supernatant is transferred in the grournd glass centrifuge tube, add 8g sodium chloride, mixing adds 25mL isopropyl alcohol+ethyl acetate (volume ratio 40+60), places oscillation extraction 20min on the oscillator.Extraction finishes, and places 5min (once centrifugal slightly as if emulsion layer is arranged).Carefully upper organic phase is moved in the rotary evaporation bottle with suction pipe, usefulness 20mL isopropyl alcohol+ethyl acetate (40+60) re-extract more once merges organic phase, is concentrated near doing in 60 ℃ on rotary evaporator.With the abundant dissolution residual substance of 1mL pH7.4 10mmol/LPBS damping fluid.Through syringe-type micro porous filtration membrane filtration, be transferred to fully in the 5mL glass centrifuge tube after washing three times, and be settled to scale with pH7.4 10mmol/LPBS damping fluid, standby.
Get the CBL-OVA lath, the sample that adds the CBL standard of 50 μ l or handle well is in micropore separately, each standard and sample must use new suction nozzle, CBL antibody 50 μ l with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 25 ℃ vibrated 1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 25 ℃ vibrated 1 hour, washed six times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.CBL content from the typical curve calculation sample sees Table 1 and Fig. 2, and CBL concentration is 0.09ng/ml in this routine sample.
Table 1
| The CBL standard point | |||||||
| CB concentration (ng/ml) | 0 | 0.05 | 0.25 | 1 | 5 | 25 | The liver sample |
| Fluorescent value (cps) | 1584332 | 1422259 | 1296643 | 1029912 | 724388 | 364570 | 1388136 |
The preparation of CBL-BSA antigen: with embodiment 1.
The preparation of polyclone antibody of clenbuteral hydrochloride: with embodiment 1.
Eu
3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 1ml that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/L Na that contains 0.155mol/L NaCl
2CO
3-NaHCO
3The pH9.0 damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.Get 500 μ l and add the Eu that contains 0.2mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 28 ℃ of magnetic agitation reactions 16 hours.(1 * 40cm) chromatography is collected protein peak to reactant liquor, and the dilution packing is standby through the Sephadex-G50 post with 80mmol/L Tris-HClpH7.8 damping fluid balance.
Bag is prepared by the plate solid phase antigen: with example 1.
The preparation of reagent:
1. clenobuterol hydrochloride standard: 0ng/ml, 0.05ng/ml, 0.25g/ml, 1ng/ml, 5ng/ml, 25ng/ml.
2. damping fluid: 8mmol/L NaCl, mass concentration 0.1% gelatin, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and contain mass concentration 0.1%NaN
350mmol/L Tris-HCl pH7.8
3. cleansing solution: 14.5mmol/L NaCl, 0.2ml/L Tween-20 and contain mass concentration 0.2%NaN
350mmol/L Tris-HCl pH7.8.
4. strengthen the preparation of liquid: 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-NTA, 50 μ mol TOPO, 1ml Triton X-100.
The reagent that kit provides
Reagent in each box enough carries out 48 measurements, and the material in the box is as follows:
(1) .1 * 48 orifice plates (4 * 12 hole can be split as single hole) are coated with CBL-OVA.
(2) .6 * CBL titer, the 1.0ml/ bottle, concentration of standard solution is: 0,0.05,0.25,1,5,25ng/ml.
(3) .1 * CBL antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
(4) .1 * EU
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
(5) .1 * enhancing liquid: 15ml.
(6) .1 * cleansing solution: 30ml, the time spent is with distilled water dilution in 1: 25.
(7) .1 * damping fluid: 30ml.
The preparation of CBL-BSA antigen: 6mgCBL is dissolved among the HCl of 100mmol/L precooling, slowly drips the 1mol/L sodium nitrite solution, checks when test paper becomes darkviolet with the KI starch paper to stop, and obtains the diazotising derivant.Taking by weighing 20mgBSA is dissolved in the carbonate buffer solution of pH9.650mmol/L.Diazotizing CBL is slowly splashed in the BSA solution, regulate pH with 1mol/LNaOH and maintain 9.0～9.5, after dropping finishes, continue reaction 4 hours, PBS with pH7.410mmol/L in 4 ℃ of refrigerators dialysed two days, obtained the CBL-BSA bond and carried out the UV scanning detection, and qualified back packing is standby.
The preparation of polyclone antibody of clenbuteral hydrochloride is identical with embodiment 1, slightly.
Eu
3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 2ml that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/L Na that contains 0.155mol/L NaCl
2CO
3-NaHCO
3The pH8.5 damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.Get 1000 μ l and add the Eu that contains 0.4mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through Sepharose CL-6B post (1 * 40cm) chromatography, A with 80mmol/L Tris-HClpH7.8 damping fluid balance
280Protein peak is collected in monitoring, and the dilution packing is standby.
The solid phase antigen preparation is identical with embodiment 1.
The preparation of reagent is identical with embodiment 1.
The reagent that kit provides is identical with embodiment 1.
The reagent that the laboratory should be provided for oneself is identical with embodiment 1.
Points for attention are with embodiment 1 before measuring.
The concrete step that detects is with embodiment 1.
Embodiment 4
The reagent that kit provides is identical with embodiment 1, is used to detect muscle samples.
Concrete detection step is as follows:
Earlier sample is handled: take by weighing muscle samples 10g (being accurate to 0.01g), with the homogenate of 20mL0.1mol/L perchloric acid solution, place the grournd glass centrifuge tube, place the ultrasonic 20min of ultrasonic cleaner then, taking-up places 80 ℃ of water-baths to heat 30min, takes out cooling back centrifugal (4500r/min) 15min.Inclining supernatant, and precipitation is with the washing of 5mL0.1mol/L perchloric acid solution, and is centrifugal again, and twice supernatant is merged.With 1mol/L sodium hydroxide solution adjust pH to 9.5 ± 0.1, if there is precipitation to produce, centrifugal again (4500r/min) 10min, supernatant is transferred in the grournd glass centrifuge tube, add 8g sodium chloride, mixing adds 25mL isopropyl alcohol+ethyl acetate (40+60), places vibration extraction 20min on the oscillator.Extraction finishes, and places 5min (once centrifugal slightly as if emulsion layer is arranged).Carefully upper organic phase is moved in the rotary evaporation bottle with suction pipe, usefulness 20mL isopropyl alcohol+ethyl acetate (40+60) re-extract more once merges organic phase, is concentrated near doing in 60 ℃ on rotary evaporator.With the abundant dissolution residual substance of 1mL pH7.4 0.01mol/LPBS damping fluid.Through syringe-type micro porous filtration membrane filtration, be transferred to fully in the 5mL glass centrifuge tube after washing three times, and be settled to scale with pH7.4 0.01mol/LPBS damping fluid, standby.
Get the CBL-OVA lath, the sample that adds the CBL standard of 50 μ l or handle well is in micropore separately, each standard and sample must use new suction nozzle, CBL antibody 50 μ l with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 37 ℃ vibrated 0.5 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 37 ℃ vibrated 0.5 hour, washed six times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.CBL content from the typical curve calculation sample sees Table 2 and Fig. 2, and CBL concentration is .0.03ng/ml in this routine sample.
Table 2
| The CBL standard point | |||||||
| CBL concentration (ng/ml) | 0 | 0.05 | 0.25 | 1 | 5 | 25 | Muscle samples |
| Fluorescent value (cps) | 1584332 | 1422259 | 1296643 | 1029912 | 724388 | 364570 | 1442482 |
The reagent that kit provides is identical with embodiment 1, is used to detect blood sample.
Concrete detection step is as follows:
Earlier blood sample is handled: get the PBS that 0.5ml blood adds 0.5mlpH7.4 10mmol/L and dilute, standby.
Get the CBL-OVA lath, the sample that adds the CBL standard of 50 μ l or handle well is in micropore separately, each standard and sample must use new suction nozzle, CBL antibody 50 μ l with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 25 ℃ vibrated 1 hour, and cleansing solution is washed 3 times, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 37 ℃ vibrated 0.5 hour, washed 6 times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.CBL content from the typical curve calculation sample sees Table 3 and Fig. 2, and CBL concentration is 2.01ng/ml in this routine sample.
Table 3
| The CBL standard point | |||||||
| CBL concentration (ng/ml) | 0 | 0.05 | 0.25 | 1 | 5 | 25 | Blood sample |
| Fluorescent value (cps) | 1584332 | 1422259 | 1296643 | 1029912 | 724388 | 364570 | 906071 |
The reagent that kit provides is identical with embodiment 1, is used to detect urine sample.
Concrete detection step is as follows:
Earlier urine sample is handled: limpid pig urine is diluted more than 10 times with the PBS of pH7.4 10mmol/L, and is standby.If the urine sample muddiness must be filtered or be centrifugal, get filtrate or supernatant and dilute back standby.
Get the CBL-OVA lath, the sample that adds the CBL standard of 50 μ l or handle well is in micropore separately, each standard and sample must use new suction nozzle, CBL antibody 50 μ l with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 25 ℃ vibrated 1 hour, and cleansing solution is washed 3 times, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 37 ℃ vibrated 0.5 hour, washed 6 times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.CBL content from the typical curve calculation sample sees Table 4 and Fig. 2, and CBL concentration is 0.47ng/ml in this routine sample.
Table 4
| The CBL standard point | |||||||
| CBL concentration (ng/ml) | 0 | 0.05 | 0.25 | 1 | 5 | 25 | Urine sample |
| Fluorescent value (cps) | 1584332 | 1422259 | 1296643 | 1029912 | 724388 | 364570 | 1183804 |
Claims (4)
1. time resolved fluoro-immunoassay method kit that detects clenobuterol hydrochloride, it is characterized in that by 96 or 48 holes bag by plate (1), damping fluid (2), clenobuterol hydrochloride standard (3), the antibody dried frozen aquatic products (4) of clenobuterol hydrochloride, the goat anti-rabbit antibody of europium mark (5), cleansing solution (6) and enhancing liquid (7) are formed;
Bag is by plate (1): bag is by solid phase antigen, with 50mmol/L pH9.6 Na
2CO
3-NaHCO
3Damping fluid clenobuterol hydrochloride-ovalbumin is diluted to 1mg/L as coating buffer, 96 or 48 each hole of hole microwell plate add 100 μ l, 4 ℃ of placements are spent the night, discard coating buffer, wash three times, add the damping fluid sealing of Tris-HCl that 200 μ l contain the 50mmol/LpH7.2 of mass concentration 0.2% gelatin, 4 ℃ of placements are spent the night, discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing;
Damping fluid (2): 8mmol/L NaCl, mass concentration 0.1% gelatin, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1ml/L Tween-80 and contain mass concentration 0.1%NaN
350mmol/L Tris-HCl pH7.8;
Clenobuterol hydrochloride standard (3): totally 6 bottles, clenobuterol hydrochloride concentration is respectively: 0ng/ml, and 0.05ng/ml, 0.25ng/ml, 1ng/ml, 5ng/ml, 25ng/ml, dilution are the PBS of pH7.4 10mmol/L;
The antibody dried frozen aquatic products (4) of clenobuterol hydrochloride: with Freund's complete adjuvant or incomplete Freund 1.2ml mixing 2mg clenobuterol hydrochloride-bovine serum albumin(BSA), mixed 2 hours with homogenizer, make Water-In-Oil antigen emulsifying agent, get the Water-In-Oil antigen emulsifying agent that 600 μ l prepare, multidigit point ground carries out hypodermic injection on one's body new zealand white rabbit, after immunity 3～4 times, serum identifies, qualified back dilution, packing, freeze-drying are standby;
The goat anti-rabbit antibody of europium mark (5): get the 5g/L goat anti-rabbit antibody 1-2ml that is dissolved in 50mmol/L PBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/LNa that contains 0.155mol/L NaCl
2CO
3-NaHCO
3The pH8.5-9.0 damping fluid is collected protein peak, and is quantitative through the uv absorption analysis, and to 2g/L, the goat anti-rabbit antibody of getting after 500-1000 μ l dilutes adds the Eu that contains 0.2-0.4mg with above-mentioned eluent dilution goat anti-rabbit antibody
3+-N
2In the bottle of-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, 28-30 ℃ of magnetic agitation reacted 16-20 hour, reactant liquor is collected protein peak through the 1 * 40cmSephadex-G50 column chromatography with 80mmol/L Tris-HCl pH7.8 damping fluid balance, and dilution, packing are standby;
Cleansing solution (6): 14.5mmol/L NaCl, 0.2ml/L Tween-80 and contain mass concentration 0.2%NaN
350mmol/L Tris-HCl pH7.8;
Strengthen liquid (7): 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1ml triton x-100.
2. method that detects clenobuterol hydrochloride with the described kit of claim 1, it is characterized in that getting be coated with clenobuterol hydrochloride-ovalbumin the micropore bag by plate, the sample that adds the clenobuterol hydrochloride standard or handled well is in micropore separately, add antibody of clenbuteral hydrochloride again, oscillating reactions, the cleansing solution washing, the goat anti-rabbit antibody that adds the europium mark, carry out the labelled immune reaction, the cleansing solution washing, add and strengthen liquid vibration back measurement fluorescence intensity cps, the content of clenobuterol hydrochloride in the reference standard curve calculation sample.
3. the method for detection clenobuterol hydrochloride according to claim 2, it is characterized in that operating process is: get be coated with clenobuterol hydrochloride-ovalbumin the micropore bag by plate, the sample that adds the clenobuterol hydrochloride standard of 50 μ l or handled well is in micropore separately, add with damping fluid (2) and make thinning agent, 50 μ l antibody of clenbuteral hydrochloride after diluting 20 times at 1: 20,25-37 ℃ vibrated 0.5-1 hour, cleansing solution is given a baby a bath on the third day after its birth time, adds with 100 μ l EU after 20 times of damping fluid (2) dilutions in 1: 20
3+-goat anti-rabbit antibody, 25-37 ℃ vibrated 0.5-1 hour, washed six times with cleansing solution, added the vibration of 200 μ l enhancing liquid and measured fluorescence intensity cps, the content of clenobuterol hydrochloride in the reference standard curve calculation sample after 5 minutes.
4. according to the method for claim 2 or 3 described detection clenobuterol hydrochlorides, it is characterized in that sample preparation wherein, animal tissue's sample preparation: with reference to GB/T5009.192-2003; Blood sample is handled: gets the PBS that 0.5ml blood adds 0.5mlpH7.410mmol/L and dilutes, and standby; Urine sample is handled: limpid pig urine sample is diluted more than 10 times with the PBS of pH7.410mmol/L, and is standby, if the urine sample muddiness must be filtered or be centrifugal, gets filtrate or supernatant and dilutes back standby.
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|---|---|---|---|
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