CN102608317A - Kit or test strips for detecting salbutamol - Google Patents
Kit or test strips for detecting salbutamol Download PDFInfo
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- CN102608317A CN102608317A CN2012100442968A CN201210044296A CN102608317A CN 102608317 A CN102608317 A CN 102608317A CN 2012100442968 A CN2012100442968 A CN 2012100442968A CN 201210044296 A CN201210044296 A CN 201210044296A CN 102608317 A CN102608317 A CN 102608317A
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Abstract
The invention discloses a kit or test strips for detecting salbutamol. The enzyme linked immunoassay kit or the test strips for detecting salbutamol comprises monoclonal antibodies secreted by salbutamol monoclonal antibody hybridoma 2E9, wherein the preservation number of the salbutamol monoclonal antibody hybridoma 2E9 is CGMCC No.5791. The kit and the test strips of the invention have the advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection time, suitability for various departments, simple storage and long quality guarantee period, and by using the kit and the test strips, samples in a large scale can be quickly detected simultaneously, and high-flux and quick detection can be realized on spot. The enzyme linked immunoassay kit and the test strips provided by the invention are suitable for assaying the residual amount of salbutamol in animal tissues, feed and veterinary drugs. The antibody, the kit and the test strips of the invention play an important role in detecting salbutamol.
Description
Technical field
The present invention relates to detect the kit or the test strips of salbutamol.
Background technology
Salbutamol (Salbutamol) is a kind of selectivity broxaterol (beta-stimulants), is used to treat the bronchial spasm due to asthma type brochitis, bronchial astehma, the pulmonary emphysema in the medical treatment.This medicine is not a veterinary drug, neither feed addictive.Because the relatively lagging behind of salbutamol detection means, but have same growth promotion effect, thereby progressively replace Clenbuterol and become a new generation's " clenbuterol hydrochloride ", be used for illegally seeking economic interests with Clenbuterol.Salbutamol adds in the feed as the heavy partitioning agent of nutrition, can promote the animal meat growth, reduce fat and produce, promote unit-economy and be worth, but also can cause drug accumulation simultaneously, remain in the animal body.There is multiple toxic and side effect in the disposable intake of salbutamol to human body more for a long time; Can cause aggregate concentration rising in the body and the secondary cardiomegaly, and cause heartbeat to overrun, myocardial infarction can occur when serious; Long-term excess intake salbutamol possibly produce reproductive endocrine toxicity.The World Health Organization (WHO), the U.S., European Union and some developed countries etc. all legislation successively forbid in Production of Livestock and Poultry, using beta-stimulants as actuating thing growth feed addictive.China has clearly stipulated to ban use of salbutamol in No. 235 bulletin of the Ministry of Agriculture, and must not in all animal foods, detect.
At present, in national standard and the industry standard liquid chromatography-mass spectrography/mass spectroscopy is often adopted in the detection of salbutamolum residue in feed and the animal derived food.Though the method high specificity, highly sensitive, the sample pre-treatments complex operation step, cost is higher, and the screening that also is not suitable for batch samples detects.Immunoassay; Since special advantages aspect the qualitative, quantitative of antigen-antibody and easy and simple to handle fast, cost is low, sensitivity is higher, the analyzing samples amount is big advantage remedied the deficiency of physico-chemical analysis, the important effect of play more and more in the residue detection of beta-stimulants.The advantage that quick detection test paper is easy and simple to handle fast, cost is low, sensitivity is higher, the analyzing samples amount is big has remedied the deficiency of physico-chemical analysis, the important effect of play more and more in the residue detection of animal tissue, feed, veterinary drug.
Summary of the invention
The purpose of this invention is to provide detect the salbutamol kit or test strips.
The enzyme linked immunological kit of detection salbutamol provided by the invention comprises the monoclonal antibody that desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 secretes.
Desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9; Be called for short hybridoma 2E9; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5791.
Said kit can be following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen.
Formula (I).
The conjugate of compound and carrier protein shown in the formula (I) is specifically suc as formula shown in (II).
Formula (II).
Said carrier protein can be ovalbumin (OVA) or bovine serum albumin(BSA) (BSA).
In the said conjugate, the coupling ratio of compound and carrier protein specifically can be (8-11) shown in the formula (I): 1.Said coupling ratio refers to mol ratio.Compound and said carrier protein can be with the carbonnitrogen bond couplings shown in the formula (I).
Said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
Per 1 liter of cleansing solution can obtain according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain cleansing solution.Said phosphate buffer can be the phosphate buffer of pH7.2-7.6,0.005M-0.015M, and the concentration that specifically can be can be pH7.4,0.01M) sodium phosphate buffer.
Said sample concentration liquid can be the phosphate buffer that concentration is 0.03mol/L-0.05mol/L, is preferably the PBS damping fluid of pH7.4,0.04mol/L.
Said substrate colour developing liquid comprises colour developing liquid A and colour developing liquid B, can be the colour developing liquid A and colour developing liquid B of independent packaging, also can directly colour developing liquid A be mixed obtaining with colour developing liquid B equal-volume.Said colour developing liquid can be superoxol or urea peroxide solution; Said colour developing liquid B can be o-phenylenediamine (OPD) solution or tetramethyl benzidine (TMB) solution.Said colour developing liquid A specifically can be 2% (g/100ml) urea peroxide WS.Said colour developing liquid B specifically can be 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
Said stop buffer specifically can be the 0.2M aqueous sulfuric acid.
More than arbitrary said kit all can be used for detecting salbutamol.
More than arbitrary said kit all can be used for detecting whether contain salbutamol in the sample to be tested.
The present invention also protects a kind of colloidal gold strip that detects salbutamol, is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads; Along test strips axially; Said absorption of sample pad, said collaurum pad, said reaction film and said adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad; The end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with the monoclonal antibody of colloid gold label on the said collaurum pad; Said monoclonal antibody is the monoclonal antibody of desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 secretion;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone (T line) is and the axial vertical ribbon of said test paper with Quality Control district (C line); Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone is coated with the conjugate (coating antigen) of compound shown in the formula (I) and carrier protein, and it is anti-that the Quality Control district encapsulates sheep anti mouse two.
Said absorption of sample pad is a cellulose filter membrane.Said collaurum pad is the glass fibre membrane that is coated with the said monoclonal antibody of colloid gold label.Said reaction film is nitrocellulose filter (a NC film).Said adsorptive pads is a thieving paper.
Said sample well is positioned on the sample absorbent away from the terminal end of collaurum pad.
Said colloidal gold strip can be used for detecting salbutamol.
Said colloidal gold strip can be used for detecting whether contain salbutamol in the sample to be tested.
The present invention adopts the salbutamol monoclonal antibody of high specific, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Kit of the present invention, test strips and detection method are low to the pre-treatment requirement of sample, and sample pretreatment process is simple, simultaneously the fast detecting batch samples.That kit of the present invention and test strips (colloidal gold test paper card) have is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long shelf-life; The fast detecting batch samples can realize on-the-spot high flux fast detecting simultaneously.Kit provided by the invention and test strips are applicable to the residual quantity of measuring salbutamol in animal tissue, feed, the veterinary drug.Antibody of the present invention, kit, test strips and detection method will be brought into play significant role in the detection of salbutamol.
Description of drawings
Fig. 1 is the ultraviolet spectrogram of salbutamol artificial antigen.
The canonical plotting of Fig. 2 for adopting salbutamol to make.
Fig. 3 is the canonical plotting of kit.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is conventional method.Used test material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used PBS damping fluid among the embodiment like no specified otherwise, is the PBS damping fluid of pH7.4,0.01M.Used carbonate buffer solution is the sodium carbonate buffer of pH9.6,0.05mol/L among the embodiment.Bovine serum albumin(BSA) is called for short BSA.Ovalbumin is called for short OVA.
Salbutamol is available from Sigma-Aldrich company, and catalog number is S8260.
Ethylene glycol diglycidylether is suc as formula shown in (III), and proportion is 1.08, and molecular weight is 158.20.
Salbutamol is suc as formula shown in (IV), and molecular weight is 239.31.
Embodiment 1, preparation salbutamol haptens
One, the haptenic preparation of salbutamol
1, take by weighing salbutamol 10mg (42 μ mol), place the 10mL Erlenmeyer flask, adding 5mL concentration is the sodium bicarbonate aqueous solution of 3% (mass ratio); Fully stirring dissolves it fully; Add 6.2 μ L (42 μ mol) ethylene glycol diglycidylether, stirring at room reaction 4h obtains solution I.
2, with the mixed liquor vacuum drying of step 1, the solid that obtains is product.
Two, the haptenic sign of salbutamol
Product to the step 1 preparation carries out ultimate analysis, and the result is following:
C：63.88；H：8.58；N：3.26；O：24.57。
The result shows that the product of step 1 preparation is a compound shown in the formula (I).
Formula (I).
Embodiment 2, preparation salbutamol artificial antigen
One, immunogenic preparation of salbutamol and sign
1, the immunogenic preparation of salbutamol
Get compound shown in the formula (I) that 10mg embodiment 1 prepares; To wherein dropwise adding bovine serum albumin solution (the 50mg bovine serum albumin(BSA) is dissolved in 5mL PBS damping fluid), continue to stir the bag filter of packing into after 8 hours, 72 hours (water is changed 6 times in the centre) of 4 ℃ of dialysis in the PBS damping fluid; Then in the centrifugal 30min of 8000rmp; Get supernatant (salbutamol immunogen solution), be sub-packed in the ampere bottle-20 ℃ of preservations.The salbutamol immunogene is called for short SAL-BSA.The salbutamol immunogen solution is called for short SAL-BSA solution.
SAL-BSA solution with after the PBS damping fluid dilution, is measured the spectrophotometric value of 280nm and 260nm, by formula calculate the concentration of the albumen in the dilution, be the SAL-BSA concentration in the SAL-BSA solution after multiply by extension rate.Protein concentration (mg/ml)=1.45 * OD
280-0.74 * OD
260SAL-BSA concentration in the SAL-BSA solution is 8.7mg/ml.
2, the immunogenic sign of salbutamol
SAL-BSA solution is diluted (concentration that makes SAL-BSA is 5mg/mL) with the PBS damping fluid, as the solution first; The PBS damping fluid that will contain the 5mg/mL salbutamol is as solution second; The PBS damping fluid that will contain 5mg/mL BSA is as solution third.Respectively solution first, solution second and solution third are carried out ultraviolet (200-380nm) spectral scan, the uv scan result sees Fig. 1.Significant change has taken place in the uv-spectrogram of comparing the solution first with solution third, and compound and BSA success coupling is described.
The maximum absorption wave long value of solution second is 282nm, and the maximum absorption wave long value of solution third is 280nm.Calculate the extinction coefficient (K) of each compound according to formula K=A/CL (A is the absorbance under the maximum absorption wave long value, and C is a solution concentration, and L is the thickness of liquid layer).
Adopt the maximum absorption wave long value of solution second and solution third that the solution first is carried out uv scan respectively; And according to the concentration of this compound of extinction coefficient backwards calculation in the solution first of this compound that has calculated; Obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value; Calculate coupling ratio, the coupling ratio of compound and BSA is 11: 1 shown in the formula (I), and promptly compound shown in 11 formulas (I) combines 1 BSA.
Two, the preparation of salbutamol coating antigen
1, the preparation of salbutamol coating antigen
Replace bovine serum albumin(BSA) with ovalbumin, other is with 1 of step 1.
The salbutamol coating antigen is called for short SAL-OVA.Salbutamol coating antigen solution is called for short SAL-OVA solution.
SAL-OVA concentration in the SAL-OVA solution is 5.6mg/ml.
2, the sign of salbutamol envelope antigen
Replace SAL-BSA with SAL-OVA, replace BSA with OVA, other is with 2 of step 1.
Significant change has taken place in the uv-spectrogram of comparing the dilution of SAL-OVA solution with OVA solution and SAL solution, and compound and OVA success coupling is described.
The coupling ratio of compound and OVA is 8: 1 shown in the formula (I), and promptly compound shown in 8 formulas (I) combines 1 OVA.
Embodiment 3, salbutamol MONOCLONAL ANTIBODIES SPECIFIC FOR
Balb/c mouse: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: available from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
SAL-BSA solution immunity Balb/c mouse with embodiment 2 preparations; Every mouse single immunization 100 μ gSAL-BSA, immunity is 4 times altogether, each two weeks at interval; The immunization ways of first three time is the subcutaneous multi-point injection of nape portion, and last immunization ways is an intraperitoneal injection.
Two, Fusion of Cells and cloning
1, the 4th immunity be after 3 days, and extracting spleen cell merges in 5: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.
2, utilize limiting dilution assay that cloning is carried out in positive hole, obtain the hybridoma that a strain can be secreted the salbutamol monoclonal antibody, called after desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 (being called for short hybridoma 2E9).Hybridoma 2E9 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012, and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5791.
Three, cell cryopreservation and recovery
With cryopreserving liquid hybridoma 2E9 is processed 1 * 10
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.During recovery, take out frozen pipe, put into 37 ℃ of water-bath middling speeds immediately and melt, move in the culture flask behind the centrifugal removal cryopreserving liquid and cultivate.
Four, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
1, increment cultivation
The preparation method of cell culture medium (7.4): in the RPMI-1640 nutrient culture media, add calf serum and soda mint, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of soda mint is 0.2% (quality percentage composition).
2E9 places cell culture medium with hybridoma, cultivates 2 days for 37 ℃, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtains monoclonal antibody (20 ℃ of preservations).
Protein concentration in the monoclonal antibody (mg/ml)=1.45 * OD
280-0.74 * OD
260
Adopt above formula to calculate the protein concentration in the monoclonal antibody, be 24.6mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilization paraffinum liquidum (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma 2E9 (5 * 10
5Individual/only).Gather ascites after 7 days, carry out purifying, the ascites behind the purifying-20 ℃ preservation with sad-saturated ammonium sulfate method.
Five, the evaluation of monoclonal antibody
The monoclonal anti liquid solution that 1 of step 4 obtains is identified respectively as follows:
1, adopt ELISA monoclonal antibody hypotype detection kit (Sigma Company products, catalog number are 19285) to detect the hypotype of monoclonal antibody, the immunoglobulin subclass of monoclonal antibody is IgG1.
2, utilize noncompetitive ELISA method to measure the affinity of monoclonal antibody
(1) with SAL-OVA as the coating antigen coated elisa plate
Adopt the SAL-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; Following SAL-OVA is set respectively encapsulates concentration: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds the dilution (diluting with the PBS damping fluid) of 100 μ L monoclonal anti liquid solutions; Protein concentration in the dilution is respectively 1.25,0.625,0.3125,1.5625 * 10
-1, 7.8 * 10
-2, 3.9 * 10
-2, 1.95 * 10
-2, 9.75 * 10
-3, 4.88 * 10
-3, 2.44 * 10
-3, 1.22 * 10
-3, 6.1 * 10
-4Mg/L; Every kind of dilution is provided with three multiple holes.
(5) incubated at room 2h washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB colour developing liquid, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
Natural logarithm value with the protein concentration in the monoclonal antibody (mol/L) is a horizontal ordinate, is that ordinate is made curve with its corresponding absorbance.
Each antigen coated concentration obtains 1 S type curve, obtains 4 S type curves altogether.Find out the top of S curve, corresponding OD
450Value is set at ODMAX.Find out the corresponding AC of each bar curve 50%ODMAX respectively.Adopt 1 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 3.4 * 10
-12Mol/L.Adopt 0.5 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 25.6 * 10
-12Mol/L.Adopt 0.25 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 170.6 * 10
-12Mol/L.Adopt 0.125 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 390.1 * 10
-12Mol/L.
With one group in twos of 4 concentration, calculate the affinity costant of monoclonal antibody according to formula
Ka＝(n-1)/2(n[Ab]t
1-[Ab]t
2)
In the formula, two multiples that encapsulate concentration during n is every group, [Ab] t
1, [Ab] t
2Be respectively two ACs (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 3.4 * 10
-12Mol/L, 0.5 μ g/mL encapsulates concentration, 50%OD
450Corresponding AC is 25.6 * 10
-12Mol/L, Ka=(2-1)/2 (2 * 25.6 * 10
-12-3.4 * 10
-12)=10.4 * 10
9M
-1And the like, obtain all the other 5 Ka values, be respectively 1.58 * 10
9M
-1, 0.82 * 10
9M
-1, 2.21 * 10
9M
-1, 0.97 * 10
9M
-1, 1.12 * 10
9M
-1, the affinity costant that calculates monoclonal antibody of averaging is 2.84 * 10
9M
-1
3, monoclonal antibody Sensitivity calculation
(1) adopt the SAL-OVA solution (diluting) of embodiment 2 preparations to encapsulate 100 μ L/ holes with carbonate buffer solution; The concentration that encapsulates of SAL-OVA is 1.0 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds 50 μ L salbutamol standard solutions and (is made up of salbutamol and PBS damping fluid; The concentration of salbutamol is respectively 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L; With the hole that only adds the PBS damping fluid as control wells), each concentration is provided with 3 multiple holes.
(5) every hole adds the monoclonal anti liquid solution that 1 of 50 μ L step 4 obtain.
(6) incubated at room 2h washes plate.
(7) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(8) wash plate.
(9) add TMB colour developing liquid, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
The light absorption value (mean values in three multiple holes) that the standard solution that adopts each concentration is obtained multiply by 100 as ordinate again divided by the light absorption value of control wells; Natural logarithm value with the salbutamol concentration in each standard solution (μ g/L) is horizontal ordinate curve plotting figure, sees Fig. 2.
Map 2 obtains Y value and equals 50% o'clock corresponding salbutamol concentration and be IC
50Value.Monoclonal antibody detects the sensitivity (IC of salbutamol
50Value) is 2.5 μ g/mL.
Embodiment 4, salbutamol Polyclonal Antibody Preparation
New zealand white rabbit: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd..
New zealand white rabbit is carried out immunity (immunization ways is the subcutaneous multi-point injection of nape portion) with the salbutamol immunogene (SAL-BSA) of preparation among the embodiment 2.Every each immune 1.5mg of rabbit (in the BSA amount); Per three all immunity once; During immunity for the first time salbutamol immunogene and Freund's complete adjuvant are mixed and made into emulsifying agent and carry out immunity, to the 6th immunity salbutamol immunogene and incomplete Freund are mixed and made into emulsifying agent for the second time and carry out immunity, the 7th immunity carried out immunity with the salbutamol immunogene; The 7th immunity be the heart blood sampling after 10 days, obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
The preparation of the enzyme linked immunological kit of embodiment 5, detection salbutamol
One, the composition of enzyme linked immunological kit
Enzyme linked immunological kit is made up of following substances:
1, cleansing solution: per 1 liter of cleansing solution obtains according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer (pH7.4,0.01M) are mixed, obtain cleansing solution.
2, encapsulate the ELISA Plate of SAL-OVA
With the SAL-OVA solution dilution (concentration that makes SAL-OVA be 0.5 μ g/mL) of carbonate buffer solution, be coating buffer with embodiment 2 preparations; Coating buffer is added 96 hole polystyrene ELISA Plates (48 holes also can), every hole 100 μ L, 37 ℃ of incubation 2h; The coating buffer that inclines, with cleansing solution washing 3 times, each 30s claps and does; In every hole, add 200 μ L confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Per 1 liter of said confining liquid is prepared according to following method: 5ml horse serum, 1g sodium azide and 30g casein are dissolved with phosphate buffer (pH7.2,0.02M) and be settled to 1000ml, obtain confining liquid.
3, sample concentration liquid: PBS damping fluid (pH7.4,0.04M).
Sample concentration liquid is diluted with water to 20 times of volumes, is sample diluting liquid.
4, antibody working fluid: the monoclonal anti liquid solution that 1 of the step 4 of embodiment 3 is obtained dilutes with sample diluting liquid, and making protein concentration is 7.0ng/mL.
5, ELIAS secondary antibody working fluid
The sheep anti-mouse antibody of horseradish peroxidase (HRP) mark is available from U.S. Sigma-Aldrich company, and catalog number is A7058, by specification preparation ELIAS secondary antibody working fluid.
6, standard solution
Standard items are that salbutamol is (available from U.S. Sigma-Aldrich company; Catalog number is S8260).
With sample diluting liquid dilution dissolving salbutamol, obtain each standard solution.Salbutamol concentration is respectively 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L in each standard solution.
Sample diluting liquid is the standard solution (0 standard) of 0 μ g/L as salbutamol concentration.
7, substrate colour developing liquid
Mixed by A liquid and B liquid equal-volume, A liquid is 2% (g/100ml) urea peroxide WS, and B liquid is 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
8, stop buffer: 0.2M aqueous sulfuric acid.
Two, kit test method
Adopt the kit of step 1 preparation to detect as follows:
(1) sample pre-treatments
The detection sample is a urine sample: get sample to be checked in centrifuge tube, add isopyknic sample diluting liquid, filter (or the centrifugal 5min of 4000g), get 20 μ L supernatants as sample to be tested solution.
Detect sample and be tissue: accurately take by weighing sample behind the homogeneous in centrifuge tube, add sample diluting liquid, fully whirling motion disperses to organizing fully, and centrifugal 10min more than the 4000g gets 20 μ L supernatants as sample to be tested solution.
The detection sample is a feed: accurately take by weighing sample behind the homogeneous in centrifuge tube, add the 0.05M HCl WS, fully whirling motion 1min; Add 0.05M NaOH WS adjust pH to 7-8; Fully whirling motion 30s leaves standstill 5min, gets 20 μ L supernatants as sample to be tested solution.
(2) kit test method
1, the making of typical curve
In the ELISA Plate that encapsulates SAL-OVA, add standard solution (20 μ L/ holes; Each standard solution is provided with three multiple holes), add ELIAS secondary antibody working fluid (50 μ L/ hole) again, add antibody working fluid (80 μ L/ hole) again; With cover plate film shrouding; React 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, (step of each washing is: every hole adds 250 μ L cleansing solutions to wash 4 times; Pour out liquid in the hole behind the 30s), clap dried with thieving paper.Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, and 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD with ELIASA
630And OD
450Dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
The absorbance mean value (B) of the standard solution of each concentration of usefulness multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtains the percentage absorbance.Utilization Originpro 7.0 softwares are analyzed the data result, are the X axle with the natural logarithm value of standard items concentration (μ g/L), and the percentage absorbance simulates typical curve for the Y axle.Typical curve is seen Fig. 3.
2, the mensuration of salbutamol concentration in the sample
In the ELISA Plate that encapsulates SAL-OVA, add sample to be tested solution or its dilution (20 μ L/ holes; Three multiple holes are set), add ELIAS secondary antibody working fluid (50 μ L/ hole) again, add antibody working fluid (80 μ L/ hole) again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, wash 4 times (step is the same), clap with thieving paper and do.Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, and 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD with ELIASA
630And OD
450Dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
The result judges: use each sample to be tested solution absorbency mean value (B) to multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtain the percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution then can be read the concentration value of salbutamol from typical curve, multiply by the extension rate of respective sample again, converses the content of salbutamol in the sample to be tested solution.
Three, kit detects effect assessment
(1) accuracy and precision test
In the urine sample that does not contain salbutamol, add salbutamol standard items (standard items), make the final concentration of salbutamol in sample be respectively 0.5 μ g/L, 1.0 μ g/L, 2 μ g/L; Sample after adding is carried out pre-treatment according to method described in () of step 2 respectively, obtain sample to be tested solution.
From the kit of three different batches, respectively extracting 3 kits detects; (2) of detection method such as step 22 described in; Each experiment repetition 5 times, according to the content of salbutamol in the cubage detection sample of salbutamol in the sample to be tested solution, the result sees table 1.
Table 1 is used the content (μ g/L) that each kit detects salbutamol in the sample that draws
| Kit 1 | Kit 2 | Kit 3 | |
| The final concentration of salbutamol in sample is 0.5 μ g/L | ?0.47 | ?0.43 | ?0.40 |
| The final concentration of salbutamol in sample is 1.0 μ g/L | ?0.82 | ?0.76 | ?0.88 |
| The final concentration of salbutamol in sample is 2.0 μ g/L | ?1.52 | ?1.67 | ?1.85 |
The difference calculate recovery rate and the coefficient of variation, the result sees table 2.
Content * 100% of the actual salbutamol that adds in the content ÷ sample of the salbutamol that the recovery=application kit detection computations goes out.
The coefficient of variation (CV)=mensuration result's the standard deviation and the number percent of its mean value.
The computing method of plate within variance coefficient: certain sample (being generally medium level) replication number of times gained result's the coefficient of variation in plate within variance coefficient=same same block of plate of once measuring.
The computing method of variation within batch coefficient: the variation within batch coefficient=coefficient of variation of each parallel samples in once measuring together.
The computing method of interassay coefficient of variation: interassay coefficient of variation=same sample is got its mean value in different batches mensuration result's the coefficient of variation.
Table 2 recovery and coefficient of variation result
The result shows: 76.0%～94.0%, the variation within batch coefficient is 4.3%～8.6% in the interpolation recovery of all samples for the recovery of all samples, and interassay coefficient of variation is 12.8%～15.1%.
(2) kit storage life
The kit preservation condition is 2-8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, salbutamol added the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur that kit the condition held of 37 ℃ of preservations 8 days, is carried out accelerated deterioration and tests, and the result shows that each item index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 8 days, measure the result and show that also kit each item index is normal fully.Can draw kit from above result can preserve more than 12 months at 2-8 ℃ at least.
(3) cross reacting rate test
In the ELISA Plate that encapsulates SAL-OVA, adding the analogue standard solution (is made up of analogue and PBS damping fluid; The concentration of analogue is respectively 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L; With the hole that only adds the PBS damping fluid as control wells; 20 μ L/ holes; Each concentration is provided with 3 multiple holes), add ELIAS secondary antibody working fluid (50 μ L/ hole) again, add antibody working fluid (80 μ L/ hole) again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, wash 4 times (step is the same), clap with thieving paper and do.Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, and 37 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD with ELIASA
630And OD
450Dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength).
With the cross reacting rate of computes kit to other analogue.
The result sees table 3.
The specificity of table 3 kit
| Medicine name | Available from Business Name and products thereof catalog number (Cat.No.) | Cross reacting rate |
| Salbutamol | U.S. Sigma-Aldrich company; Catalog number is S8260 | 100％ |
| Clenbuterol | U.S. Sigma-Aldrich company; Catalog number is 54969 | ＜1％ |
| Ractopamine | U.S. Sigma-Aldrich company; Catalog number is 34198 | ＜1％ |
| Gram rumba amine | U.S. Sigma-Aldrich company; Catalog number does | ＜1％ |
| Terbutaline | U.S. Sigma-Aldrich company; Catalog number is T2528 | ＜1％ |
| Wei Duoluoer | U.S. Sigma-Aldrich company; Catalog number is N1892 | ＜1％ |
Experiment shows, kit of the present invention only produces reaction with salbutamol, and do not produce cross reaction with analog Clenbuterol, Ractopamine, Ke Lunba amine, Terbutaline and the Wei Duoluoer of salbutamol, explains that this kit has good specificity.
Embodiment 6, the test strips that detects salbutamol and preparation and application
One, the structure of test strips
Said test strips is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially; Absorption of sample pad, collaurum pad, reaction film and adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad, and the end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with 1 monoclonal antibody that obtains of step 4 of the embodiment 3 of colloid gold label on the said collaurum pad;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone (T line) is and the axial vertical ribbon of test strips with Quality Control district (C line); Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone encapsulates the SAL-OVA of embodiment 2 preparations, and it is anti-that the Quality Control district encapsulates sheep anti mouse two.
Sample well is positioned on the sample absorbent away from the terminal end of collaurum pad.
Two, the preparation of test strips
(1) colloid gold label antibody
1. the preparation of colloidal gold solution
Get 0.01% aqueous solution of chloraurate 100mL and be heated to boiling with the constant temperature magnetic stirrer, continue to add 1% trisodium citrate aqueous solution 2.5mL under the condition of stirring, continue agitating heating 20min, solution is bright redness.The room temperature cooling returns to original volume with deionized water, 2-8 ℃ of preservation.
The preparation of 2. golden labeling antibody solution
Use 0.1mol/L K
2CO
3The WS is regulated the pH to 8.2 of colloidal gold solution; Getting 10mL then adds in the 50mL beaker; Magnetic stirrer 250r/min stirs; Dropwise add the 1 monoclonal anti liquid solution (containing 0.35mg protein) that obtains of the step 4 of embodiment 3, dropwise add the 3mL 5g/100mL BSA WS, continue to stir 10min.
3. with the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500r/min), discard the deposition that forms by the gold grain that condenses, get red supernatant solution.
4. with 4 ℃ of step solution 3., the centrifugal 40min of 11000r/min; Solution is divided into three layers (fine and close gold grain layers of black on transparent supernatant, the flowable kermesinus deposition in the pipe end and the pipe diapire); Flowable kermesinus deposition is transferred in the another one centrifuge tube, used the 0.01mol/L phosphate buffer that contains 1g/100mL BSA to be suspended to the volume of former golden labeling antibody solution, spend the night; 4 ℃, the centrifugal 40min of 11000r/min, collecting precipitation.
5. with the 0.01mol/L phosphate buffer that contains 1g/100mL BSA and 0.02g/100mL NaN3 step deposition 4. is suspended to former golden labeling antibody solution volume 1/40,2-8 ℃ of preservation.
(2) metal spraying: the suspension that step (1) is obtained is sprayed onto on the glass fibre membrane, processes the collaurum pad.
(3) spray film: the T line position on reaction film is sprayed SAL-OVA solution, the C line position of embodiment 2 preparations and is sprayed sheep anti-mouse antibody.
(4) assembling: absorption of sample pad (cellulose filter membrane), collaurum pad, nitrocellulose filter (NC film), adsorptive pads (thieving paper) are assembled by conventional method, and slitting is then packed test strips in the plastics fabrication into, forms test card.
Three, detect with test card
(1) sample pre-treatments and detection
(one) with the step 2 of embodiment 5.
Take out test card, lie against desktop behind the Kaifeng, draw sample to be tested solution and dropwise add 4 in sample well; The 5-10min judged result, the judged result behind the 15min is invalid.
Criterion as a result:
Negative: the colour developing of C line, T line naked eyes are visible, and no matter shade all is judged to feminine gender;
Positive: the colour developing of C line, the T line does not develop the color, and is judged to the positive;
Invalid: the C line does not develop the color, and no matter whether the T line develops the color, and it is invalid that this test card all is judged to.
Four, the effect of test card
(1) false positive rate and false negative rate
50 parts of the negative urines (containing salbutamol＜3 μ g/kg) of the conclusive evidence of learning from else's experience, 50 parts of the positive urines (containing salbutamol >=3 μ g/kg) of the conclusive evidence of learning from else's experience.Sample is detected with test card respectively, calculate false positive rate and false positive rate.
The result: in 50 parts of negative urine samples, test card detects totally 1 part of positive, and false positive rate is 2%.In 50 parts of positive urine sample determinations, test card detects 0 part of negative sample, and false negative rate is 0%.
(2) test card storage life
Stability test is the result show, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.
Claims (7)
1. an enzyme linked immunological kit that detects salbutamol comprises the monoclonal antibody that desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 secretes; The deposit number of said desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 is CGMCC No.5791.
2. kit as claimed in claim 1 is characterized in that: said kit is following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen;
Formula (I).
3. kit as claimed in claim 2 is characterized in that: said carrier protein is ovalbumin or bovine serum albumin(BSA).
4. like arbitrary described kit in the claim 1 to 3, it is characterized in that: said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
5. a colloidal gold strip that detects salbutamol is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially; Said absorption of sample pad, said collaurum pad, said reaction film and said adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad; The end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with the monoclonal antibody of colloid gold label on the said collaurum pad; Said monoclonal antibody is the monoclonal antibody of desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 secretion; The deposit number of said desertification butylamine alcohol monoclonal antibody hybridoma cell 2E9 is CGMCC No.5791;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone is and the axial vertical ribbon of said test paper with the Quality Control district; Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone is coated with the conjugate of compound shown in the formula (I) and carrier protein, and it is anti-that the Quality Control district encapsulates sheep anti mouse two;
Formula (I).
6. arbitrary said kit in the claim 1 to 4, or, the said colloidal gold strip of claim 5, the application in detecting salbutamol.
7. arbitrary said kit in the claim 1 to 4, or whether the said colloidal gold strip of claim 5 contains the application in the salbutamol detecting sample to be tested.
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| CN201210044296.8A CN102608317B (en) | 2012-02-23 | 2012-02-23 | Kit or test strips for detecting salbutamol |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103343109A (en) * | 2013-07-24 | 2013-10-09 | 泰州康正生物技术有限公司 | Hybridoma cell strain, anti-salbutamol monoclonal antibody generated by hybridoma cell strain and application |
| CN105628929A (en) * | 2014-11-25 | 2016-06-01 | 北京维德维康生物技术有限公司 | Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines |
| CN110004117A (en) * | 2019-04-11 | 2019-07-12 | 中抗生物医药(杭州)有限公司 | Secrete hybridoma cell strain, monoclonal antibody and the application of salbutamol monoclonal antibody |
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| CN105628929A (en) * | 2014-11-25 | 2016-06-01 | 北京维德维康生物技术有限公司 | Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines |
| CN110004117A (en) * | 2019-04-11 | 2019-07-12 | 中抗生物医药(杭州)有限公司 | Secrete hybridoma cell strain, monoclonal antibody and the application of salbutamol monoclonal antibody |
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