CN102262157A - Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby - Google Patents
Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby Download PDFInfo
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- CN102262157A CN102262157A CN2011101892159A CN201110189215A CN102262157A CN 102262157 A CN102262157 A CN 102262157A CN 2011101892159 A CN2011101892159 A CN 2011101892159A CN 201110189215 A CN201110189215 A CN 201110189215A CN 102262157 A CN102262157 A CN 102262157A
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Abstract
The invention discloses a quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and a special kit thereby. The immunofluorescence kit for detecting the clenbuterol hydrochloride, which is provided by the invention, comprises a clenbuterol hydrochloride coating antigen and a quantum dot-marked clenbuterol hydrochlorid antibody. The quantum dot-based method disclosed by the invention can be used for quantitatively detecting a residual medicament of the clenbuterol hydrochloride and is also suitable for detecting various samples as well as has the advantages of low detectability, high detection sensitivity and favorable specificity. Therefore, the quantum dot-based method has a wide application prospect in the field of detection of the clenbuterol hydrochlorid.
Description
Technical field
The present invention relates to a kind of method and dedicated kit that detects clenobuterol hydrochloride based on the immunofluorescence of quantum dot.
Background technology
Clenizole Hydrochloride (Clenbuterol, CL) belong to beta-stimulants, illegally added to lean meat percentage and the acceleration growth of animal to improve the lard type animal in the feed in recent years, because of its additive capacity be therapeutic dose 5-10 doubly, to such an extent as to residual quantity is high and bring harm to the consumer in animal body.At present, the goldstandard that is used for clenobuterol hydrochloride residue thing detection by quantitative is chromatogram/mass spectroscopy, but its sample pretreatment process is loaded down with trivial details time-consuming, the testing cost height.ELISA method based on competitiveness enzyme-linked immune response principle need not carried out complicated sample pre-treatment process, and detection time is short relatively, but its precision is not enough, can only be used for qualitatively screening, can't be used for quantitative conclusive evidence.The sample size that detects clenobuterol hydrochloride residue and pollution situation investigation thereof in the current livestock products is big, and task is heavy, and cost is high, and sense cycle is long.Therefore, be badly in need of stable, quick, the economic standard method of testing result.
(Quantum Dots, QDs) marker material is the class new material that development in recent years is got up to quantum dot, comprises II-VI family and III-V family semiconductor nano.Because the luminous and absorption characteristic that quantum dot is outstanding, make its have fluorescence lifetime length and width excitation spectrum, narrow emission spectrum, can be accurately tuning emission wavelength, very high photochemical stability, can carry out advantageous characteristic such as multi-color marking, adopt its material that serves as a mark, can realize the ultramicron of target molecules is detected.
Requirements such as quantum yield height, fluorescence are strong because the quantum dot-labeled material that is applicable to immune diagnostic reagent must satisfy, good biocompatibility, highly stable and cost are low, and existing external its quantum yield of business-like quantum dot is many below 40%, its size is less than normal, need shine with laser optical apparatus and excite, only can be applied in cellular immunofluorescence detection and aspects such as fluidic cell detection and sorting at present, so also not have the quantum dot immune detectable to come out on the international market.
Summary of the invention
An object of the present invention is to provide a kind of immunofluorescence detection agent box that is used to detect clenobuterol hydrochloride.
The immunofluorescence detection agent box that is used to detect clenobuterol hydrochloride provided by the present invention comprises clenobuterol hydrochloride coating antigen and quantum dot-labeled antibody of clenbuteral hydrochloride.
In the mentioned reagent box, described antibody of clenbuteral hydrochloride is that antibody titer is 10
6More than (be specially 10
6), the antibody affinity costant is 10
6～10
8M
-1Or 10
7～10
8M
-1Or 10
8M
-1The clenobuterol hydrochloride monoclonal antibody; Being specially antibody titer is 10
6, the antibody affinity costant is 10
8M
-1The clenobuterol hydrochloride monoclonal antibody, described clenobuterol hydrochloride monoclonal antibody is available from Beijing Sheng Dilong bio tech ltd, catalog number is CL-083.
Described quantum dot is that surperficial carboxyl-content is 1 * 10
-3～9 * 10
-3Mmol/mg or 1 * 10
-3～6 * 10
-3Mmol/mg or 5 * 10
-3The quantum dot of the water-soluble CdSe of mmol/mg/ZnS nucleocapsid structure; The quantum yield of described quantum dot is 40～70% or 50～70% or 60%; The excitation wavelength of described quantum dot is 345nm, and emission wavelength is 620nm.
In above-mentioned arbitrary described kit, the particle diameter of described quantum dot is 10～20nm, and described particle diameter is specially 13～20nm, and described particle diameter especially is preferably 20nm; The deviation of its particle diameter (CV) is specially between 10～20% between 10～30%, is specially 15% again;
In above-mentioned arbitrary described kit, in the described quantum dot-labeled antibody of clenbuteral hydrochloride, the amino on carboxyl on the described quantum dot and the described antibody of clenbuteral hydrochloride forms peptide bond, and then described quantum dot is connected with described antibody of clenbuteral hydrochloride.
In above-mentioned arbitrary described kit, described clenobuterol hydrochloride coating antigen is the conjugate of clenobuterol hydrochloride and carrier protein, and wherein said clenobuterol hydrochloride and described carrier protein are connected by the peptide bond that carboxyl in the clenobuterol hydrochloride and amino in the carrier protein form; Described carrier protein is any in the gamma globulin of bovine serum albumin(BSA), human serum albumins, keyhole limpet hemocyanin, thyroglobulin, albumin rabbit serum, ovalbumin, fibrinogen and rabbit and chicken.
In above-mentioned arbitrary described kit, also comprise clenobuterol hydrochloride standard items, dilution, cleansing solution in the described kit, contain porose polystyrene board, bag is cushioned liquid and confining liquid;
Described clenobuterol hydrochloride standard items are 4-amino-α-(tert-butylamine methyl)-3,5-dichlorbenzyl alcohol hydrochloride;
Described dilution is the PBS damping fluid; Be specially the PBS damping fluid of 0.02M, pH7.4.
Described cleansing solution is the PBST damping fluid; Specifically be prepared as follows: the NaN3 that gets 0.2ml Tween20 and 0.1g is dissolved in the described dilution, and the dissolving back is settled to 1L with dilution.
It is carbonate buffer solution that described bag is cushioned liquid; Be specially the carbonate buffer solution of 0.05M, pH9.6.
Described confining liquid is the PBS damping fluid that contains the albumen that is useful on the bag quilt; The described albumen that is used for wrapping quilt is any of BSA, ovalbumin and hemocyanin.Specifically be prepared as follows: 10g BSA and 0.2mlTween20 are dissolved in the above-mentioned dilution, and the dissolving back is settled to 1L with dilution.
In above-mentioned arbitrary described kit, 0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L described standard items are the standard items of following solution form: with described 4-amino-α-(tert-butylamine methyl)-3,5-dichlorbenzyl alcohol hydrochloride is diluted to the solution of following each concentration with described dilution:.
In above-mentioned arbitrary described kit, described clenobuterol hydrochloride coating antigen is coated on described containing on the porose polystyrene board, method for coating dilutes the coating buffer that described clenobuterol hydrochloride coating antigen obtains 10 μ g/ml for being cushioned liquid with described bag, adds 100 μ l in every hole.
In above-mentioned arbitrary described kit, described quantum dot-labeled antibody of clenbuteral hydrochloride is present in the kit with following solution form: the solution that the described quantum dot-labeled antibody of clenbuteral hydrochloride of every 25ug is obtained with the described diluted of 5ml.
Another object of the present invention provides the method for clenobuterol hydrochloride in a kind of test sample.
The method of clenobuterol hydrochloride in the test sample provided by the present invention comprises the steps: with above-mentioned arbitrary described kit testing sample to be detected, and described testing sample is animal muscle tissue's sample, animal urine sample or feed.Described testing sample is specially pig urine.
Detection side's ratio juris of the present invention: adopt quantum dot as the fluorescence signal labeled molecule, with clenobuterol hydrochloride antigen direct coated in the micropore of polystyrene board, add clenobuterol hydrochloride standard items or test sample, and quantum dot-labeled antibody of clenbuteral hydrochloride, make it form Ag-Ab binary electrochemiluminescent immunoassay compound, excite and detect the fluorescence intensity of this immunofluorescence compound with fluorescence detector, by with measure the concentration that the typical curve contrast that forms obtains clenobuterol hydrochloride to be measured.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled antibody of clenbuteral hydrochloride, the clenobuterol hydrochloride antigen of bag quilt combines antibody of clenbuteral hydrochloride competitively with clenobuterol hydrochloride in the test sample, and the specificity by Ag-Ab is in conjunction with formation Ag-Ab binary immune complex.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled antibody of clenbuteral hydrochloride and test sample simultaneously, the clenobuterol hydrochloride antigen that is coated in the polystyrene micropore combines with antibody of clenbuteral hydrochloride competitively with clenobuterol hydrochloride in the test sample, forms the Ag-Ab binary immune complex that is fixed in the microwell plate wherein combine remaining antibody of clenbuteral hydrochloride and the clenobuterol hydrochloride antigen generation specific bond that is coated in the microwell plate with test sample after.
Clenobuterol hydrochloride immunofluorescence detection agent of the present invention is relevant with quantum dot-labeled immunofluorescence detection technique, be to adopt quantum dot as the fluorescence signal marker material, carry out class methods of immunofluorescence quantitative measurement, this technology has been integrated the research of association areas such as the chemosynthesis of fluorescence quantum nano material, finishing and labelling technique, indirect competition formula immunoassay technology.
Why the present invention can detect clenobuterol hydrochloride, be to adopt a kind of method based on quantum dot-labeled immunofluorescence quantitative measurement, be about to clenobuterol hydrochloride antigen direct coated in the micropore of polystyrene board, measuring principle based on quantum dot-labeled indirect competition immunofluorescence assay, adding clenobuterol hydrochloride standard items or test sample, and behind the quantum dot-labeled antibody of clenbuteral hydrochloride, the fluorescence intensity that is attached to the Ag-Ab binary immune complex in the polystyrene board micropore by detection realizes the detection to clenobuterol hydrochloride: be attached to the quantity difference of the quantum dot-labeled antibody of polystyrene board micropore, the fluorescence intensity that is produced is also different.The content of the clenobuterol hydrochloride in the finite concentration scope in the height of fluorescence intensity level and the sample is inversely proportional to.Can be made into typical curve by the clenobuterol hydrochloride standard items that add variable concentrations, the fluorescence intensity level of inquiring about each test sample according to this typical curve can obtain corresponding clenobuterol hydrochloride drug concentrations value.
Its concrete technical step comprises:
(1) preparation of fluorescence quantum point mark probe: the water soluble fluorescence quantum dot that adopt to be fit to, activate its surperficial carboxyl after, adopt the mode of chemical coupling that the antibody of clenbuteral hydrochloride orientation is connected to the quantum dot surface.
(2) envelope antigen: adopt clenobuterol hydrochloride antigen with the bovine serum albumin(BSA) coupling as envelope antigen, the method by physisorption with this antigen direct coated in the polystyrene board micropore.
(3) formation of Ag-Ab fluorescence immunoassay compound: add clenobuterol hydrochloride standard items or test sample in by good polystyrene board micropore in above-mentioned bag, and quantum dot-labeled antibody of clenbuteral hydrochloride, the clenobuterol hydrochloride antigen that is adsorbed in the hole combines with antibody of clenbuteral hydrochloride with the clenobuterol hydrochloride in standard or the sample is emulative, and the specificity by Ag-Ab is in conjunction with forming Ag-Ab binary electrochemiluminescent immunoassay compound.
(4) quantitative fluorescence detects: adopt fluorescence microplate reader to excite and detect the fluorescence intensity of above-mentioned formed Ag-Ab fluorescence immunoassay compound; Excitation wavelength: 345nm; Emission wavelength: 620nm; Form typical curve by the fluorescence intensity of measuring serial corresponding standard items, by contrasting the concentration that obtains clenobuterol hydrochloride to be measured with the typical curve of measuring formation.
The formation of described Ag-Ab binary electrochemiluminescent immunoassay compound is: after adding quantum dot-labeled antibody of clenbuteral hydrochloride, the clenobuterol hydrochloride antigen of bag quilt combines antibody of clenbuteral hydrochloride competitively with the clenobuterol hydrochloride in standard or the sample, specificity by Ag-Ab is in conjunction with forming Ag-Ab binary immune complex, quantum dot of mark can fluoresce after exciting on it, the excitation wavelength that the present invention uses is 345nm, emission wavelength is 620nm, the Ag-Ab immune complex that obtains glowing.
The detection of described fluorescence intensity is the fluorescence intensity that excites and detect formed Ag-Ab binary electrochemiluminescent immunoassay compound with fluorescence microplate reader, because the antibody that is adopted is fixed concentration, usually the clenobuterol hydrochloride drug concentrations in the testing sample is high more, many more by the medication amount of antibody capture, the antibody that combines with the clenobuterol hydrochloride antigen of bag quilt is few more, and the fluorescence intensity level that records is low more.
Because quantum dot carrying out will carrying out separation and purification with ultracentrifugation in the coupling with antibody, quantum dot that particle diameter is too little such as 8nm and 10nm's can't be centrifugal, can't purifying after the coupling, and result of use is relatively poor; Quantum dot that particle diameter is too big such as the ratio more than the 60nm are easier to assemble, and it is relatively poor to be used on the kit homogeneity.
The particle diameter of described quantum dot is 10～20nm, and described particle diameter is specially 13～20nm, and described particle diameter especially is preferably 20nm; The deviation of its particle diameter (CV) is preferably between 10～20% between 10～30%, and preferred 15%.
The fluorescence quantum yield of quantum dot and fluorescence intensity thereof have directly determined the height of detection sensitivity and accuracy thereof, and the fluorescence quantum yield of the quantum dot of classic method preparation all is lower than 40% usually, fluorescence intensity a little less than.
For improving its sensitivity and accuracy, the quantum yield of described quantum dot is 40～70%, and the fluorescence quantum yield of described quantum dot is specially 50～70%, and the fluorescent quantum product of described quantum dot especially is preferably 60%.
Survey for being used for the clenobuterol hydrochloride residue quality testing, the quantum dot surface need have the group that is easy to the antibody of clenbuteral hydrochloride coupling, these groups can be carboxyl, amino groups, the group of optimizing is the surface functional group of band carboxyl, usually adopt chemical method to connect antibody, promptly, close reaction and finish coupling reaction with antibody generation carboxylic again with behind EDC and the NHS activation quantum dot.
Before the condensate with the formation of peptide bond covalent bond, also comprise the step that activates described quantum dot surface functional group with antibody of clenbuteral hydrochloride and quantum dot.The carboxyl-content difference on quantum dot surface can have influence on the sensitivity of detection, and for improving sensitivity, described functional group is specially carboxyl, and the content of described carboxyl is 1 * 10
-3～9 * 10
-3Mmol/mg, the content of described carboxyl is specially 1 * 10
-3～6 * 10
-3Mmol/mg, the content of described carboxyl especially is preferably 5 * 10
-3Mmol/mg.
In immune detection, the performance index of antibody are most important for the accuracy that detects, and usually, high specificity, the antibody that affinity is high can improve the accuracy of detection significantly.Discover that for improving sensitivity, described antibody of clenbuteral hydrochloride affinity costant is 10
6～10
8M
-1Described antibody of clenbuteral hydrochloride affinity costant is specially 10
7～10
8M
-1Described antibody of clenbuteral hydrochloride affinity costant especially is preferably 10
8M
-1
Because clenobuterol hydrochloride is a small-molecule substance, its molecular surface characteristic is unfavorable for combining with the direct of polystyrene micropore plate, itself and carrier protein need be carried out could reaching by means of the character of surface of carrier protein after the coupling good combination with polystyrene micropore plate.The seralbumin that various animals are arranged that can be used as carrier protein, as bovine serum albumin(BSA) (Bovine SerumAlbumin, BSA), human serum albumins (Human SerumAlbumin, HSA), also has keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH), thyroglobulin, albumin rabbit serum (RSA), ovalbumin (Ovalbumin, OVA), the gamma globulin of fibrinogen or rabbit and chicken.Discover, the BSA physicochemical property is stable, the lysine content height, free amino group is many, bigger solubleness is all arranged under different pH and ionic strength, under the situation that contains organic solvent (as pyridine, DMF etc.), all can carry out coupling, and after coupling, still keep solvable state with haptens, be splendid selection, so the present invention selects for use BSA as coupling protein as carrier protein.
The present invention is by the research to fluorescence quantum, clenobuterol hydrochloride antigen and antibody of clenbuteral hydrochloride molecular characterization, by optimization to the preparation of various water soluble fluorescence quantum dot, coating and finishing condition, water soluble fluorescence quantum dot and the specific antibody selecting to be fit to carry out directed covalent chemical coupling, obtain functional fluorescence quantum point mark probe, and, reach quick and highly sensitive quantitative measurement to the clenobuterol hydrochloride residue medicine by optimizing the various conditions of competitive immunization reaction.Experiment showed, kit of the present invention highly sensitive, specificity good, accuracy is high.The inventive method can realize the detection by quantitative to the clenobuterol hydrochloride residue medicine, and detectability is low, detection sensitivity is high, specificity is good, also is applicable to the detection of several samples.Therefore, the present invention has broad application prospects at the detection range of clenobuterol hydrochloride.
Description of drawings
Fig. 1 water-soluble CdSe/ZnS fluorescence quantum Electronic Speculum (TEM) photo.
Fig. 2 detects the typical curve of clenobuterol hydrochloride with quantum dot-labeled indirect competition immunofluorescence technique.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The composition of embodiment 1, detection kit and preparation
Clenobuterol hydrochloride opens a day chemical industry Institute for Research and Technology, catalog number: 30229CDCT-C11668550 available from the permanent unit in Beijing.Its chemical name is 4-amino-α-(tert-butylamine methyl)-3,5-dichlorbenzyl alcohol hydrochloride.
1, clenobuterol hydrochloride coating antigen
The synthetic employing diazotising method of CL-BSA antigen, concrete steps are as follows:
(1) get in the HCl solution that 4mg clenobuterol hydrochloride (CL) is dissolved in 1ml 0.2mol/l, fully the dissolving back adds 8mg NaNO
2, in 4 ℃ of abundant down reaction 3h, this moment, solution became the diazobenzene hydrochloride that yellow is a Clenbuterol; Reaction pH value is 1;
(2) at 0.02mol/l, pH adds the BSA-PBS solution that 40mg BSA is mixed with 5mg/ml among 7.4 the PBS;
(3) add BSA-PBS solution 8ml in the above-mentioned diazobenzene hydrochloride solution for preparing of 1ml, being adjusted to pH with 2mol/lNaOH is 8.5, carries out coupling reaction 6h under 4 ℃, obtains crocus solution;
(4) reactant liquor is in 0.02mol/l, and pH is the 24h that dialyses among 7.4 the PBS, and every 6h changes a dislysate.In-20 ℃ of preservations, obtain clenobuterol hydrochloride antigen after the freeze-drying of products obtained therefrom usefulness freeze dryer.
2, the bag quilt of clenobuterol hydrochloride coating antigen
Adopt bag to be cushioned liquid the dilution of clenobuterol hydrochloride coating antigen is the coating buffer of concentration 10 μ g/ml, every hole adds 100 μ l in 96 hole polystyrene micropore laths, places in 4 ℃ of refrigerators and spends the night.After discarding coating buffer in second day, washing each plate hole 3 times with cleansing solution, each hole adds 200 μ l confining liquids, handles 2h in 37 ℃ of sealings.After discarding confining liquid afterwards, washing each plate hole 3 times with cleansing solution, carry out vacuum drain, with being positioned over-20 ℃ of preservations after the aluminium foil bag sealing.
3, quantum dot-labeled antibody of clenbuteral hydrochloride:
Antibody of clenbuteral hydrochloride is 10 for tiring
6, affinity costant is 10
8M
-1The clenobuterol hydrochloride monoclonal antibody, it is available from Beijing Sheng Dilong bio tech ltd, catalog number is CL-083.
Quantum dot is available from Shenzhen's TELUS Science and Technology Ltd., and catalog number is
LumiQD
TM20.The sign of this quantum dot is as follows: particle diameter is that the CV of 20nm, particle diameter is 15%, and quantum yield is 60%, and surperficial carboxyl-content is 5 * 10
-3Mmol/mg, water-soluble, CdSe/ZnS nucleocapsid structure, excitation wavelength are 345nm, emission wavelength is 620nm; The red fluorescence quantum dot.The scintigram of quantum dot as shown in Figure 2.
In the described quantum dot-labeled antibody of clenbuteral hydrochloride, the amino on carboxyl on the described quantum dot and the described antibody of clenbuteral hydrochloride forms peptide bond, and then described quantum dot is connected with described antibody of clenbuteral hydrochloride.
Quantum dot marking method is:
1) get the above-mentioned quantum dot of 2.5mg with the MES damping fluid of 0.1M (take by weighing 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, transfer pH to 4.7) washing and remove supernatant with the 20000rpm centrifugal enrichment after, with 1ml concentration is that 0.1M, pH value are that 4.7 MES damping fluid is resuspended, adds 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) of 0.96mg (final concentration is 5mM) and 1.15mg (final concentration is 10mM) N-maloyl imines (NHS) in wherein.Temperature of reaction is 37 ℃, the reaction half an hour after, the quantum dot after obtaining activating;
2), get the borate buffer solution that 0.15mg clenobuterol hydrochloride monoclonal antibody and 2.5mg activation back quantum dot is mixed into 0.8ml 50mM pH=8.5 and (take by weighing 1.9gNa with the borate buffer solution washing of 50mM pH=8.5
2B
4O
7.10H
2O is dissolved in the 100ml pure water, transfers pH to 8.5) in abundant mixing.Down reaction 3.5 hours of room temperature (25 ℃) allows antibody and quantum dot form stable peptide bond covalent bond, obtains containing the reactant liquor of quantum dot after the coupling;
3) after reaction finishes, to step 2) to add final concentration in the reactant liquor that obtains be the BSA (Sigma-Aldrich of 5% (quality percentage composition), 85041C) the residual activity amino sites is sealed, be reflected at and carried out under 37 ℃ 0.5 hour, obtain containing the reactant liquor of sealing back quantum dot; After finishing, (take by weighing 2.3g Na with the 0.02M PBS damping fluid of pH=7.4
2HPO
4, 0.524g NaH
2PO
4.H
2O, 8.77g NaCL are dissolved in the 1L pure water, transfer pH to 7.4) washing, the 20000rpm centrifugal enrichment is removed supernatant, and resuspended back 4 ℃ of preservations are stand-by, obtain quantum dot-labeled antibody of clenbuteral hydrochloride.
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L 4, clenobuterol hydrochloride standard items: the solution that clenobuterol hydrochloride is diluted to following each concentration with dilution:;
5, the PBS damping fluid of dilution: 0.02M, pH7.4;
Preparation: take by weighing 2.3g Na
2HPO
4, 0.524g NaH
2PO
4.H
2O and 8.77g NaCL are dissolved in the 1L pure water, transfer pH to 7.4.
6, cleansing solution: PBST damping fluid; Get the NaN of 0.2ml Tween20 and 0.1g
3Be dissolved in the above-mentioned PBS damping fluid, the dissolving back is settled to 1L with above-mentioned PBS damping fluid.
7, bag is cushioned the carbonate buffer solution of liquid: 0.05M, pH9.6.
8, confining liquid: 10g BSA and 0.2ml Tween20 are dissolved in the above-mentioned PBS damping fluid, and the dissolving back is settled to 1L with the PBS damping fluid.
The preparation method of embodiment 2, typical curve
In the clenobuterol hydrochloride micropore lath for preparing, add concentration and be 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5, the clenobuterol hydrochloride standard solution of 10ug/L, the 50ul/ hole, quantum dot-labeled ENR antibody is diluted [being the 25ug labelled antibody: the 5ml dilution] with the PBS-T dilution at 1: 50, add 50ul in each micropore, room temperature vibration 1 hour detects its fluorescence intensity numerical value with fluorescence microplate reader after cleansing solution is washed 3 times.Fluorescence microplate reader is set to excitation wavelength 345nm, emission wavelength 620nm.
The mass concentration of the competition medicine of (B0 is 0 standard sample detected value, and B is for treating the test sample detected value) was determined the sensitivity of detection architecture when the detectability of competing detection was decided to be B0/B=1.2 according to Regression Equations.Testing result is as shown in table 1 below, and detection limit is decided to be 0.001ug/L.
The quantum dot kit detected value of table 1 clenobuterol hydrochloride variable concentrations sample
The mensuration of embodiment 3, cross reaction
(a day chemical industry Institute for Research and Technology is opened by the permanent unit in Beijing to select salbutamol, 30252CDCT-C16903000), (a day chemical industry Institute for Research and Technology is opened to bricalin by the permanent unit in Beijing, 13384NIC-100273), (a day chemical industry Institute for Research and Technology is opened to isoprenaline hydrochloride by the permanent unit in Beijing, 13287NIC-100166), (a day chemical industry Institute for Research and Technology is opened to Ractopamine by the permanent unit in Beijing, 30230CDCT-C16805000) (a day chemical industry Institute for Research and Technology is opened by the permanent unit in Beijing with special sieve of Zeeman, 30251CDEO-BA016) 5 kinds of medicines, be made into series concentration respectively, detect with quantum dot kit.Calculate the IC50 that respectively competes thing, calculate the cross reacting rate of these 5 kinds of medicines and clenobuterol hydrochloride quantum dot kit with following formula respectively.Computing formula is: cross reacting rate (%)=[IC50 (clenobuterol hydrochloride)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.The result show the clenobuterol hydrochloride quantum dot kit to the cross reacting rate of 5 kinds of medicines all less than 0.1%.
The cross reaction of table 2 clenobuterol hydrochloride quantum dot kit and other medicines
The mensuration of embodiment 4, accuracy
(1) sample extraction:
1, musculature sample: take by weighing muscle or liver sample 5g (being accurate to 0.01g), add the homogenate of 10ml 0.1mol/L hydrochloric acid solution, place 80 degree water-bath heating 30 minutes.Take out cooling back centrifugal (4000g) 15min.Get supernatant, transfer PH to 7-9, get 50ul and be used for detecting with the 1mol/L sodium hydroxide solution.
2, urine sample: the urine of clarification can be directly used in detection, if cloudy urine needs centrifugal (4000g) 10min earlier, gets supernatant and detects.
3, feed: take by weighing feed 3g (being accurate to 0.01g), add the hydrochloric acid solution of 2ml 0.1mol/L, mixing; Add 8ml 0.02M PBS, mixing; Place the ultrasonic 20min of ultrasonic cleaner then, take out centrifugal (4000g) 10min; Get supernatant, transfer PH to 7-9, get 50ul and be used for detecting with NaOH.
(2) mensuration of the recovery
Detect 30 parts of negative pig urine samples, wherein 5 parts of negative urine samples are added the CL titer (0.01,0.1,0.5,1,5ug/L) of variable concentrations.Add each test of sample 5 times, and calculate recovery rate.
Determination of recovery rates the results are shown in Table 3, and it is 84%～106% that clenobuterol hydrochloride adds the sample recovery, average recovery rate 94.08%, and the coefficient of variation 4.72%～9.74%, average coefficient of variation 7.55%, accuracy is better.
Table 3 determination of recovery rates
Claims (8)
1. an immunofluorescence detection agent box that is used to detect clenobuterol hydrochloride comprises clenobuterol hydrochloride coating antigen and quantum dot-labeled antibody of clenbuteral hydrochloride.
2. kit according to claim 1 is characterized in that:
Described antibody of clenbuteral hydrochloride is that antibody titer is 10
6More than, the antibody affinity costant is 10
6～10
8M
-1The clenobuterol hydrochloride monoclonal antibody;
Described quantum dot is that surperficial carboxyl-content is 1 * 10
-3～9 * 10
-3The quantum dot of the water-soluble CdSe of mmol/mg/ZnS nucleocapsid structure; The quantum yield of described quantum dot is 40～70%.
3. kit according to claim 1 and 2 is characterized in that:
The particle diameter of described quantum dot is 10～20nm, and the deviation of its particle diameter is between 10～30%;
Described clenobuterol hydrochloride coating antigen is the conjugate of clenobuterol hydrochloride and carrier protein.
4. according to arbitrary described kit among the claim 1-3, it is characterized in that: also comprise clenobuterol hydrochloride standard items, dilution, cleansing solution in the described kit, contain porose polystyrene board, bag is cushioned liquid and confining liquid;
Described clenobuterol hydrochloride standard items are 4-amino-α-(tert-butylamine methyl)-3,5-dichlorbenzyl alcohol hydrochloride;
Described dilution is the PBS damping fluid;
Described cleansing solution is the PBST damping fluid;
It is carbonate buffer solution that described bag is cushioned liquid;
Described confining liquid is the PBS damping fluid that contains the albumen that is useful on the bag quilt.
5. according to arbitrary described kit among the claim 1-4,0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L it is characterized in that: described standard items are the standard items of following solution form: with described 4-amino-α-(tert-butylamine methyl)-3,5-dichlorbenzyl alcohol hydrochloride is diluted to the solution of following each concentration with described dilution:.
6. according to arbitrary described kit among the claim 1-5, it is characterized in that: described clenobuterol hydrochloride coating antigen is coated on described containing on the porose polystyrene board, method for coating dilutes the coating buffer that described clenobuterol hydrochloride coating antigen obtains 10 μ g/ml for being cushioned liquid with described bag, adds 100 μ l in every hole.
7. according to arbitrary described kit among the claim 1-6, it is characterized in that: described quantum dot-labeled antibody of clenbuteral hydrochloride is present in the kit with following solution form: the solution that the described quantum dot-labeled antibody of clenbuteral hydrochloride of every 25ug is obtained with the described diluted of 5ml.
8. the method for clenobuterol hydrochloride in the test sample comprises the steps: with arbitrary described kit among the claim 1-7 testing sample to be detected, and described testing sample is animal muscle tissue's sample, animal urine sample or feed.
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|---|---|---|---|
| CN201110189215.9A CN102262157B (en) | 2011-07-07 | 2011-07-07 | Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby |
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| CN108152257A (en) * | 2017-12-12 | 2018-06-12 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of method that Zilpaterol is measured using CdTe quantum |
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| CN108037105A (en) * | 2017-12-12 | 2018-05-15 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of method using CdTe quantum measure Zilpaterol |
| CN108152257A (en) * | 2017-12-12 | 2018-06-12 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of method that Zilpaterol is measured using CdTe quantum |
| CN108037105B (en) * | 2017-12-12 | 2020-08-04 | 江西省农业科学院农产品质量安全与标准研究所 | Method for measuring zilpaterol by utilizing CdTe quantum dots |
| CN114264815A (en) * | 2021-12-13 | 2022-04-01 | 深圳容金科技有限公司 | Quantum dot immunoassay kit for clenbuterol content and detection method |
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| CN102262157B (en) | 2014-02-05 |
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