CN101962418B - Method for preparing clenbuterol liquid phase chip probe - Google Patents
Method for preparing clenbuterol liquid phase chip probe Download PDFInfo
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- CN101962418B CN101962418B CN 201010281365 CN201010281365A CN101962418B CN 101962418 B CN101962418 B CN 101962418B CN 201010281365 CN201010281365 CN 201010281365 CN 201010281365 A CN201010281365 A CN 201010281365A CN 101962418 B CN101962418 B CN 101962418B
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Abstract
The invention relates to a method for preparing a clenbuterol liquid phase chip probe in the technical field of immunochemistry. The method comprises the following steps of: modifying the surface of a carboxylated polystyrene fluorescent microsphere with p-aminobenzoic acid, m-aminobenzoic acid or o-aminobenzoic acid by adopting a carbodiimide method; and then marking clenbuterol molecules on the surface of the fluorescent microsphere by the carbodiimide method to finally obtain the clenbuterol liquid phase chip probe. The method for preparing the clenbuterol liquid phase chip probe has the advantages of simple operation, high stability and favorable clenbuterol targeted property of products and can provide a more convenient approach for the development of test technology of the clenbuterol liquid phase chip.
Description
Technical field
The present invention relates to the preparation method of a kind of clenbuterol detection reagent in immunochemical technique field, relate in particular to a kind of preparation method of clenbuterol liquid phase chip probe.
Background technology
Clenbuterol (Clenbuterol, CL) be a kind of synthetic beta 2-receptor hormone, can improve the lean ratio that several domestic animals comprise pig after making an addition in the feed, its mechanism of action is, clenbuterol is good absorption in domestic animal and human body, and pig feed accelerates conversion and the decomposition of fat with promote afterwards protein synthesis in metabolic process, improve the lean ratio of pork, therefore be called clenbuterol hydrochloride.Compare with other beta-stimulants, its bioavailability is high, occurs poisoning so that eaten the pork that contains clenbuterol.The edible pork liver of people or pig lung enough cause poisoning.The world was used for promoting animal growth without any positive gauge mechanism approval clenbuterol as fodder additives, and China began to forbid that clenbuterol is used for promoting animal growth as fodder additives in 2002.Residual for clenbuterol in the animal-derived food product, be main mainly with chromatograph-mass spectrometer coupling method, euzymelinked immunosorbent assay (ELISA) both at home and abroad, chromatograph-mass spectrometer coupling method detection sensitivity is high, but Laboratory Instruments and personnel are required height, detection method is complicated, it is lower to detect flux, and cost is higher, is difficult to satisfy the detection demand of great amount of samples in the reality.The euzymelinked immunosorbent assay (ELISA) testing cost is lower, but general detection of drugs kind is single, detects linear narrow range.
Liquid-phase chip technology is a kind of multi-functional analysis platform of U.S. Luminex company exploitation.It organically combines flow cytometer detection and chip technology, when keeping high throughput testing, reaction system is changed into liquid-phase reaction system by liquid phase-solid state reaction, for guaranteeing that the protein interaction on the higher structure is particularly crucial.The carrier of liquid-phase chip technology is the microballon of polystyrene made, coats ruddiness and the infrared light chromogenic reagent of different ratios, can produce the microballon of 100 kinds of different ratios colors, and each microballon that is used for label probe is all with the color numbers of a uniqueness.Each microballoon adopts the different probe mark, and microballon and the determinand of label probe reacted in liquid phase, reaction product is by the sense channel of liquid-phase chip, only allow a microballoon by detecting communication channel at every turn, excite survey by two kinds of laser inspections, red laser can be located the encode address of microballoon, and green laser then excites the reporter molecules (R-bath Lactoferrin) of sample to be tested to realize the quantitative analysis of sample object thing.The detecting pattern of liquid-phase chip can realize detecting the diversity of target compound, and high to the sample utilization ratio, detection speed is fast, can different types of probe be made up according to the needs of actual detected object, and handiness is large.
In recent years, in the liquid-phase chip system, realized detection to small-molecule drug.The common method of the preparation technology of small-molecule drug liquid-phase chip probe is: at first prepare the conjugate of small molecules haptens and carrier proteins, then with this conjugate and the reaction of carboxylated fluorescent microsphere, thereby obtain this micromolecular liquid-phase chip probe.This process using carrier proteins is connecting arm, because the reaction repeatability of carrier proteins and small molecules linked reaction and conjugate and microballoon is relatively poor, has limited the application in its reality.
Summary of the invention
The object of the invention is to propose a kind of preparation method of clenbuterol liquid phase chip probe, it adopts the mode of the fluorescent microsphere surface markers clenbuterol molecule of using at liquid-phase chip, but the Novel immune that constructs a kind of specific recognition antibody of clenbuteral is analyzed solid phase interface, its further Application and Development in the liquid-phase chip mode detection of clenbuterol, thereby overcome the deficiencies in the prior art.
For achieving the above object, the present invention has adopted following technical scheme:
A kind of preparation method of clenbuterol liquid phase chip probe is characterized in that, the method is:
(1) getting the carboxylic polystyrene fluorescent microsphere, to be dispersed in the pH value be 6.0～7.0, contain in the phosphate buffer soln of 0.05M～0.2M, after supersound process, the 50mg/ml nitrogen N-Hydroxysuccinimide sulfonate and the 50mg/ml water-soluble carbodiimide aqueous solution that add again excessive fresh configuration, 15～60min is hatched at room temperature dark place concussion, the centrifugal supernatant of removing then, the Para-Aminobenzoic that adds excessive 0.01～0.1wt%, m-anthranilic acid or ortho-, meta-or p-aminobenzoic acid solution, after 15～30min is hatched in the dark place under the room temperature, recentrifuge is removed supernatant, take the pH value as 7.2～8.0, the phosphate buffer soln washing precipitate that contains 0.05M～0.2M makes the fluorescent microsphere after the first activation;
(2) fluorescent microsphere after the above-mentioned first activation is added in the aqueous solution of the excessive freshly prepared 50mg/ml of containing nitrogen N-Hydroxysuccinimide sulfonate and 50mg/ml water-soluble carbodiimide, after evenly mixing, 15～30min is hatched in the dark place concussion under the room temperature, finish the again activation to fluorescent microsphere, add the excessive phosphate buffer soln that contains 0.01～1 μ g/mL clenbuterol thereafter, 1～2h is hatched in the dark place concussion under the room temperature, thereafter the centrifugal supernatant of removing, take the pH value as 7.2～8.0, contain the phosphate buffer soln washing precipitate of 0.05M～0.2M, make the target product clenbuterol liquid phase chip probe.
Say that further in the step (1), described carboxylic polystyrene fluorescent microsphere is after washing is processed, in the phosphate buffer soln that be scattered in again the pH value and be 6.0～7.0, concentration is 0.05M～0.2M.
In the step (1), the time of described supersound process is 1～5min, and ultrasonic frequency is 20KHz.
Described clenbuterol liquid phase chip probe is suspended in preservation in the stock solution, described stock solution is that the pH value is 7.2～8.0, concentration is the phosphate buffer soln (PBS) of 0.05M～0.2M, and it contains 0.1～1.0wt% bovine serum albumin, 0.01～0.05wt% tween 20 and 0.1wt% sodium azide.
Described phosphate buffered saline(PBS) adopts the phosphate buffered saline buffer that contains 0.8%wtNaCl.
The method is specially:
(1) get the carboxylic polystyrene fluorescent microsphere, through washing, centrifugal and remove supernatant liquor after, adding pH is 6.0～7.0, contain in the phosphate buffer soln of 0.05M～0.2M, whirlpool mixing 1min, the concentration that makes contained fluorescent microsphere in this phosphate buffer soln is 5 * 10
3～2.5 * 10
4Individual/μ L, then ultrasonic 1～5min, the 50mg/ml nitrogen N-Hydroxysuccinimide sulfonate and the 50mg/ml water-soluble carbodiimide aqueous solution that add again excessive fresh configuration, behind the whirlpool mixing 10s, 30～60min is hatched in the dark place concussion under the room temperature, the centrifugal supernatant of removing then, the Para-Aminobenzoic that adds excessive 0.01～0.1wt%, m-anthranilic acid or ortho-, meta-or p-aminobenzoic acid solution, after 15～30min is hatched in the dark place under the room temperature, the centrifugal supernatant of removing, take the pH value as 7.2～8.0, concentration is washing precipitate twice in the phosphate buffer soln of 0.05M～0.2M, makes the fluorescent microsphere after the first activation;
(2) fluorescent microsphere after will activating for the first time adds in the aqueous solution of the excessive 50mg/ml of containing nitrogen N-Hydroxysuccinimide sulfonate and 50mg/ml water-soluble carbodiimide, behind the whirlpool mixing 10s, 15～30min is hatched in the dark place concussion under room temperature, finish the re-activation of fluorescent microsphere is processed, then in the above-mentioned aqueous solution, add the excessive phosphate buffer soln formation hybrid reaction system that contains 0.01～1 μ g/mL clenbuterol, with this hybrid reaction system after 1～2h is hatched in dark place concussion under the room temperature, the centrifugal supernatant of removing, the precipitation warp is take the pH value as 7.2～8.0, concentration is the phosphate buffer soln washed twice of 0.05M～0.2M, namely make the target product clenbuterol liquid phase chip probe, this target product is suspended in the stock solution to be preserved, it is 7.2～8.0 that described stock solution adopts the pH value, concentration is the phosphate buffered saline(PBS) of 0.05M～0.2M, and it contains 0.1～1.0wt% bovine serum albumin, 0.01～0.05wt% tween 20 and 0.05～0.2wt% sodium azide.
The present invention adopts two-step approach at fluorescent microsphere surface markers clenbuterol, the first step adopts the water-soluble carbodiimide method with the amino coupled of carboxyl and the connecting arm (benzaminic acid at contraposition, a position or ortho position) of microsphere surface, second step is again with the amino coupled of method with carboxyl and the clenbuterol molecule of connecting arm, each step reaction all is directed, and the amount of microsphere surface label probe can realize control and repeat by the concentration of regulating the clenbuterol molecule.
Compared with prior art, beneficial effect of the present invention is: the easy operation of the preparation method of this clenbuterol liquid phase chip probe, controllability is strong, good reproducibility, good stability, productive rate is high, and purity is high, and product has good clenbuterol targeting, and the liquid-phase chip measuring technology exploitation that can be clenbuterol provides more convenient approach.
Description of drawings
Below in conjunction with drawings and the specific embodiments content of the present invention is described further.
Fig. 1 be among the embodiment 1 clenbuterol liquid phase chip probe to the inhibited reaction curve of clenbuterol zero standard solution;
Fig. 2 be among the embodiment 2 clenbuterol liquid phase chip probe to the inhibited reaction curve of clenbuterol zero standard solution.
Embodiment
Preparation method's principle of embodiment 1 this clenbuterol liquid phase chip probe is shown below:
In the following formula, ● be carboxylated microballoon, Sulfo-NHS is nitrogen N-Hydroxysuccinimide sulfonate, and EDC is water-soluble carbodiimide.
The method comprises following concrete steps:
(1) the finishing Para-Aminobenzoic connecting arm of fluorescent microsphere
Get 5 * 10
5Individual carboxylic polystyrene fluorescent microsphere, through washing, centrifugal and remove supernatant liquor after, add 100 μ L activation solution (0.1M phosphate buffer solns, pH 6.0), whirlpool mixing 1min, then ultrasonic 1min, 50mg/ml nitrogen N-Hydroxysuccinimide sulfonate (sulfo-NHS) and each 10 μ L of water-soluble carbodiimide (EDC.HCl) aqueous solution of adding fresh configuration, behind the whirlpool mixing 10s, 15min is hatched in the dark place concussion under the room temperature, with the centrifugal supernatant of removing of microballoon after the activation, the Para-Aminobenzoic solution 0.5mL that adds 0.01wt%, 60min is hatched in the dark place under the room temperature, the centrifugal supernatant of removing, and precipitation adopts 0.5ml phosphate buffer soln (0.1M, pH7.2) washed twice makes the fluorescent microsphere of finishing;
(2) the surface markers clenbuterol molecule of fluorescent microsphere
Sulfo-NHS and each 10 μ L of the EDC.HCl aqueous solution of adding 50mg/mL in the fluorescent microsphere after the above-mentioned modification, behind the whirlpool mixing 10s, 20min is hatched in the dark place concussion under the room temperature, again after the activation, add 450 μ l clenbuterol (0.01 μ g/mL, be dissolved in 0.1M, pH7.2 phosphate buffer soln solution), 1h is hatched in the dark place concussion under the room temperature, and the centrifugal supernatant of removing is with 0.1M, twice of the PBS solution washing precipitation of pH7.2, make the target clenbuterol liquid phase chip probe, this clenbuterol liquid phase chip probe is suspended in the stock solution to be preserved, and this stock solution is 0.1M, the PBS solution of pH7.2, it contains the 0.1wt% bovine serum albumin, 0.01wt%Tween, and 0.2wt% sodium azide).
The above phosphate buffered saline(PBS) that adopts preferably adopts the phosphate buffered saline buffer that contains 0.8%wtNaCl.
Prepared clenbuterol liquid phase chip probe is diluted a series of concentration, and then test concentrations under the microballoon count mode of Luminex 100 IS 2.2 liquid-phase chip systems is diluted to clenbuterol liquid phase chip probe about 50/μ l.Draw 200 μ l washingss (0.01M PBS, pH 7.2; Contain 0.01%Tween-20), join in the flat screen plates in 96 holes (Mi Libo),
Vacuum is drained (perhaps in the Microplate centrifuge under 130 * g centrifugal 20s) in the suction filtration system (Millipore Corp.), then in 2 row micropores, add respectively the clenbuterol zero standard solution of 50 μ L and the standardized solution of 10ng/mL, add again 10 μ l clenbuterol liquid phase chip probes (that is the haptenic fluorescent microsphere of mark), and a series of extension rate biotinylation antibody of clenbuteral (4 μ g/mL of 40 μ l, add the antibody dilution multiple by different rows and be respectively 500,1000,2000,4000,8000 and 16000), place the microwell plate vibrator, 37 ℃ of lower lucifuges are hatched 40min, vacuum is drained solution in the micropore, add 200 μ l washingss, vibration 10s, drain, repeated washing is twice again; Add 100 μ l streptavidin phycoerythrin (10 μ g/mL) thereafter, place the microwell plate vibrator, 37 ℃ of lower lucifuges are hatched 15min, then be positioned on liquid-phase chip Luminex 100 IS 2.2 system platforms, system draws coupled antigen from each hole microballoon reads 100 to I haven't seen you for ages.Fluorescence intermediate value with every kind of fluorescent microsphere of read is made the inhibited reaction curve to concentration of standard solution, the result shows that 10ng/mL clenbuterol standardized solution can be higher than 70% (seeing Fig. 1) to the association reaction inhibition level of this microballoon probe and antibody, and this shows that this clenbuterol liquid phase chip probe has very high sensitivity to clenbuterol.
And with this clenbuterol liquid phase chip probe the cross reaction of clenbuterol analog salbutamol, Ractopamine hydrochloride is tested its relatively poor reactivity of demonstration less than 0.5%, this further specifies this clenbuterol probe and has very high specific target tropism.
Preparation method's principle of embodiment 2 these clenbuterol liquid phase chip probes is shown below:
In the following formula, ● be carboxylated microballoon, Sulfo-NHS is nitrogen N-Hydroxysuccinimide sulfonate, and EDC is water-soluble carbodiimide.
The method is specific as follows:
(1) the finishing m-anthranilic acid connecting arm of fluorescent microsphere
Get 10
6Individual carboxylic polystyrene fluorescent microsphere, through washing, centrifugal and remove supernatant liquor after, add 100 μ L activation solutions (0.05M phosphate buffer soln, pH 6.5), whirlpool mixing 1min, then ultrasonic 3min.50mg/ml nitrogen N-Hydroxysuccinimide sulfonate (sulfo-NHS) and each 10 μ L of water-soluble carbodiimide (EDC.HCl) aqueous solution of adding fresh configuration, behind the whirlpool mixing 10s, 20min is hatched in the dark place concussion under the room temperature, with the centrifugal supernatant of removing of microballoon after the activation, the m-anthranilic acid solution 0.5mL that adds 0.05wt%, 60min is hatched in the dark place under the room temperature, the centrifugal supernatant of removing, precipitation adopts 0.5ml phosphate buffer soln (0.05M, pH7.6) washed twice makes the fluorescent microsphere of finishing;
(2) the surface markers clenbuterol molecule of fluorescent microsphere
Add sulfo-NHS and each 10 μ L of the EDC.HCl aqueous solution of 50mg/mL in the fluorescent microsphere after the above-mentioned modification, behind whirlpool the mixings 10s, the dark place is shaken and is hatched 20min under the room temperature.Again after the activation, add 450 μ l clenbuterols (0.1 μ g/mL is dissolved in 0.05M, the pH7.6 phosphate buffer soln), 1.5h is hatched in the dark place concussion under the room temperature.The centrifugal supernatant of removing, precipitation is with 0.5ml phosphate buffer soln (0.05M, pH7.6) washed twice, make the target clenbuterol liquid phase chip probe, this clenbuterol liquid phase chip probe is suspended in the stock solution to be preserved, and this stock solution is 0.05M, the PBS solution of pH7.5, it contains the 0.5wt% bovine serum albumin, 0.02wt%Tween, and 0.2wt% sodium azide).
The above phosphate buffered saline(PBS) that adopts preferably adopts the phosphate buffered saline buffer that contains 0.8%wtNaCl.
This clenbuterol liquid phase chip probe can detect concentration at the clenbuterol molecule (as shown in Figure 2) of 0.01ng/mL when using (its operation is similar to embodiment 1), highly sensitive, targeting is good.
The preparation method's of embodiment 3 these clenbuterol liquid phase chip probes principle is shown below:
In the following formula, ● be carboxylated microballoon, Sulfo-NHS is nitrogen N-Hydroxysuccinimide sulfonate, and EDC is water-soluble carbodiimide.
The method comprises the steps:
(1) the ortho-, meta-or p-benzaminic acid connecting arm of the finishing of fluorescent microsphere
Get 2.5 * 10
6Individual carboxylic polystyrene fluorescent microsphere, through washing, centrifugal and remove supernatant liquor after, add 100 μ L activation solutions (0.01M phosphate buffer soln, pH 7.0), whirlpool mixing 1min, then ultrasonic 5min.50mg/ml nitrogen N-Hydroxysuccinimide sulfonate (sulfo-NHS) and each 10 μ L of water-soluble carbodiimide (EDC.HCl) aqueous solution of adding fresh configuration, behind the whirlpool mixing 10s, 60min is hatched in the dark place concussion under the room temperature.With the centrifugal supernatant of removing of microballoon after the activation, add the ortho-, meta-or p-aminobenzoic acid solution 0.5mL of 0.1wt%, 45min is hatched in the dark place under the room temperature.The centrifugal supernatant of removing, precipitation adopts 0.5ml phosphate buffer soln (0.01M, pH8.0) washed twice, makes the fluorescent microsphere of finishing.
(2) the surface markers clenbuterol molecule of fluorescent microsphere
In the fluorescent microsphere after the above-mentioned modification, sulfo-NHS and each 10 μ L of the EDC.HCl aqueous solution of adding 50mg/mL, behind the whirlpool mixing 10s, 20min is hatched in the dark place concussion under the room temperature, after the activation, (1 μ g/mL is dissolved in 0.01M to add 450 μ l clenbuterols again, pH8.0 solution), 2h is hatched in the dark place concussion under the room temperature.The centrifugal supernatant that removes, precipitation is with 0.5ml phosphate buffer soln (0.01M, the pH8.0 phosphate buffered saline buffer) washed twice, make clenbuterol liquid phase chip probe, this clenbuterol liquid phase chip probe is suspended in the stock solution to be preserved, and this stock solution is 0.01M, the PBS solution of pH8.0, it contains the 1wt% bovine serum albumin, 0.05wt%Tween, and 0.2wt% sodium azide).The effect of this clenbuterol liquid phase chip probe and embodiment 1,2 close.
The above phosphate buffered saline(PBS) that adopts preferably adopts the phosphate buffered saline buffer that contains 0.8%wtNaCl.
Above embodiment only is used for illustrating content of the present invention; in addition; the present invention also has other embodiments, as long as those skilled in the art because of technology involved in the present invention enlightenment, replace or technical scheme that the equivalent deformation mode forms all drops in protection scope of the present invention and adopt to be equal to.
Claims (6)
1. the preparation method of a clenbuterol liquid phase chip probe is characterized in that, the method is:
(1) getting the carboxylic polystyrene fluorescent microsphere, to be dispersed in the pH value be 6.0～7.0,0.05M in the phosphate buffer soln of～0.2M, after supersound process, the 50mg/ml N-hydroxy-succinamide sulfonate and the 50mg/ml water-soluble carbodiimide aqueous solution that add again excessive fresh configuration, 15～60min is hatched at room temperature dark place concussion, the centrifugal supernatant of removing then, add excessive 0.01～0.1wt% Para-Aminobenzoic, m-anthranilic acid or ortho-, meta-or p-aminobenzoic acid solution, after 15～30min is hatched in the dark place under the room temperature, recentrifuge is removed supernatant, take the pH value as 7.2～8.0, concentration is the phosphate buffer soln washing precipitate of 0.05M～0.2M, makes the fluorescent microsphere after the first activation;
(2) fluorescent microsphere after the above-mentioned first activation is added in the aqueous solution of the excessive freshly prepared 50mg/ml of containing N-hydroxy-succinamide sulfonate and 50mg/ml water-soluble carbodiimide, after evenly mixing, 15～30min is hatched in the dark place concussion under the room temperature, finish the again activation to fluorescent microsphere, add the excessive phosphate buffer soln that contains 0.01～1 μ g/mL clenbuterol thereafter, 1～2h is hatched in the dark place concussion under the room temperature, thereafter the centrifugal supernatant of removing, take the pH value as 7.2～8.0, concentration is the phosphate buffer soln washing precipitate of 0.05M～0.2M, makes the target product clenbuterol liquid phase chip probe.
2. the preparation method of clenbuterol liquid phase chip probe according to claim 1, it is characterized in that: in the step (1), described carboxylic polystyrene fluorescent microsphere is after washing is processed, in the phosphate buffer soln that be scattered in again the pH value and be 6.0～7.0, concentration is 0.05M～0.2M.
3. the preparation method of clenbuterol liquid phase chip probe according to claim 1, it is characterized in that: in the step (1), the time of described supersound process is 1～5min, and ultrasonic frequency is 20KHz.
4. the preparation method of clenbuterol liquid phase chip probe according to claim 1, it is characterized in that: described clenbuterol liquid phase chip probe is suspended in preservation in the stock solution, described stock solution is that the pH value is 7.2～8.0, concentration is the phosphate buffer soln of 0.05M～0.2M, and it contains 0.1～1.0wt% bovine serum albumin, 0.01～0.05wt% tween 20 and 0.1wt% sodium azide.
5. it is characterized in that according to claim 2 or the preparation method of 4 described clenbuterol liquid phase chip probes: described phosphate buffer soln adopts the phosphate buffered saline buffer that contains 0.8wt%NaCl.
6. the preparation method of clenbuterol liquid phase chip probe according to claim 1, it is characterized in that: the method is specially:
(1) get the carboxylic polystyrene fluorescent microsphere, through washing, centrifugal and remove supernatant liquor after, adding pH is 6.0～7.0,0.05M in the phosphate buffer soln of～0.2M, whirlpool mixing 1min, the concentration that makes contained fluorescent microsphere in this phosphate buffer soln is 5 * 10
3～2.5 * 10
4Individual/μ L, then ultrasonic 1～5min, the 50mg/ml N-hydroxy-succinamide sulfonate and the 50mg/ml water-soluble carbodiimide aqueous solution that add again excessive fresh configuration, behind the whirlpool mixing 10s, 30～60min is hatched in the dark place concussion under the room temperature, the centrifugal supernatant of removing then, add excessive 0.01～0.1wt% Para-Aminobenzoic, m-anthranilic acid or ortho-, meta-or p-aminobenzoic acid solution, after 15～30min is hatched in the dark place under the room temperature, the centrifugal supernatant of removing, take the pH value as 7.2～8.0, concentration is washing precipitate twice in the phosphate buffer soln of 0.05M～0.2M, makes the fluorescent microsphere after the first activation;
(2) fluorescent microsphere after will activating for the first time adds in the aqueous solution of the excessive 50mg/ml of containing N-hydroxy-succinamide sulfonate and 50mg/ml water-soluble carbodiimide, behind the whirlpool mixing 10s, 15～30min is hatched in the dark place concussion under room temperature, finish the re-activation of fluorescent microsphere is processed, then in the above-mentioned aqueous solution, add the excessive phosphate buffer soln formation hybrid reaction system that contains 0.01～1 μ g/mL clenbuterol, with this hybrid reaction system after 1～2h is hatched in dark place concussion under the room temperature, the centrifugal supernatant of removing, take the pH value as 7.2～8.0, concentration is twice of the phosphate buffer soln washing precipitate of 0.05M～0.2M, namely make the target product clenbuterol liquid phase chip probe, this target product is suspended in the stock solution to be preserved, it is 7.2～8.0 that described stock solution adopts the pH value, concentration is the phosphate buffer soln of 0.05M～0.2M, and it contains 0.1～1.0wt% bovine serum albumin, 0.01～0.05wt% tween 20 and 0.05～0.2wt% sodium azide.
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