CN102288765B - Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit - Google Patents
Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit Download PDFInfo
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Abstract
The invention discloses an immunofluorescence chloramphenicol detecting method based on quantum dots and a special kit. The immunofluorescence detection kit used for detecting chloramphenicol provided by the invention comprises a chloramphenicol coating antigen and chloramphenicol antibodies marked by the quantum dots. The invention can realize the quantitative detection of chloramphenicol residues, and has a low detection limit, high detection sensitivity and good specificity, and is suitable for detecting a variety of samples. Therefore, the invention has broad application prospects in the field of chloramphenicol detection.
Description
Technical field
The present invention relates to a kind of method and dedicated kit of the immunofluorescence chlorine detection mycin based on quantum dot.
Background technology
Chloromycetin (Chloramphenicol, CAP) is widely used antibiotic, once livestock and poultry control and treatment has been played to vital role.But because there is serious spinoff in chloromycetin, can cause people's alpastic anemia, the diseases such as agranulocytosis, the developed countries such as America and Europe forbid or strictly forbid using in succession.The situation of abusing again and again in China for chloromycetin, China in " animal food residue of veterinary drug regulation " also the regulation edible part must not detect, and, in the routine testing of outlet, classify them as essential items for inspection, once find to exceed standard, forbid outlet.At present, the goldstandard quantitatively detected for the residual chloromycetin thing is the Chromatography/Mass Spectrometry method, but its sample pretreatment process is loaded down with trivial details time-consuming, and testing cost is high.ELISA method based on competitiveness enzyme-linked immune response principle does not need to carry out complicated sample pretreatment process, and detection time is relatively short, but its precision is inadequate, can only be for qualitatively screening, and can't be for quantitative conclusive evidence.The sample size that detects residual chloromycetin and Survey of contaminating status thereof in the agricultural product such as current outlet aquatic products is large, and the task weight spends highly, and sense cycle is long.Therefore, be badly in need of stable, quick, the economic standard method of testing result.
Quantum dot (Quantum Dots, QDs) marker material is the class new material that development in recent years is got up, and comprises II-VI family and III-V family semiconductor nano.Due to the outstanding luminous and absorption characteristic of quantum dot, but make its emission wavelength with fluorescence lifetime length and width excitation spectrum, narrow emission spectrum precision tuning, very high photochemical stability, can carry out the advantageous characteristic such as multi-color marking, adopt its material that serves as a mark, can realize the ultramicron of target molecules is detected.
Because the quantum dot-labeled material that is applicable to immune diagnostic reagent must meet that quantum yield is high, fluorescence is strong, the requirement such as good biocompatibility, highly stable and cost be low, and existing external its quantum yield of business-like quantum dot is many below 40%, its size is less than normal, need to irradiate and excite with laser optical apparatus, only can be applied at present cellular immunofluorescence detection and the aspects such as fluidic cell detection and sorting, so on international market, also not have quantum dot immune to detect the reagent appearance.
Summary of the invention
An object of the present invention is to provide a kind of box of the immunofluorescence detection agent for detection of chloromycetin.
Immunofluorescence detection agent for detection of chloromycetin box provided by the present invention, comprise chloromycetin coating antigen and quantum dot-labeled chloramphenicol antibody.
In the mentioned reagent box, described chloramphenicol antibody is that antibody titer is 10
6Abovely (be specially 10
6), the antibody affinity costant is 10
6～10
8M
-1Or 10
7～10
8M
-1Or 10
8M
-1The chloromycetin monoclonal antibody; Being specially antibody titer is 10
6, the antibody affinity costant is 10
8M
-1The chloromycetin monoclonal antibody, described chloromycetin monoclonal antibody is purchased from Yunnan University's monoclonal antibody engineering center, catalog number is CAP-001.
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 core shell 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), between 10～30%, is specially between 10～20%, then is specially 15%;
In above-mentioned arbitrary described kit, in described quantum dot-labeled chloramphenicol antibody, the amino on the carboxyl on described quantum dot and described chloramphenicol antibody forms peptide bond, and then described quantum dot is connected with described chloramphenicol antibody.
In above-mentioned arbitrary described kit, the conjugate that described chloromycetin coating antigen is chloromycetin and carrier protein, the peptide bond that wherein said chloromycetin and described carrier protein form with amino in carrier protein by the carboxyl introduced in chloromycetin is connected; Any in the gamma globulin that described carrier protein is 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 chloromycetin standard items, dilution, cleansing solution in described kit, contain porose polystyrene board, coated damping fluid and confining liquid;
Described chloromycetin standard items be D-Su Shi-(-)-N-[(-(hydroxymethyl)-(hydroxyl-p-nitrophenyl ethyl]-2, the 2-dichloro acetamide;
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 described dilution, after dissolving, with dilution, is settled to 1L.
Described coated damping fluid is carbonate buffer solution; Be specially the carbonate buffer solution of 0.05M, pH9.6.
Described confining liquid is the PBS damping fluid containing being useful on coated albumen; Described is any of BSA, ovalbumin and hemocyanin for coated albumen.Specifically be prepared as follows: 10g BSA and 0.2mlTween20 are dissolved in above-mentioned dilution, after dissolving, with dilution, are settled to 1L.
In above-mentioned arbitrary described kit, the standard items that described standard items are following solution form: with described dilution by described D-Su Shi-(-)-N-[(-(hydroxymethyl)-(hydroxyl-p-nitrophenyl ethyl]-2,0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L the 2-dichloro acetamide is diluted to the solution of following each concentration:.
In above-mentioned arbitrary described kit, described chloromycetin coating antigen is coated on described containing on porose polystyrene board, and method for coating, for described coated damping fluid, to dilute the coating buffer that described chloromycetin coating antigen obtains 10 μ g/ml, adds 100 μ l in every hole.
In above-mentioned arbitrary described kit, described quantum dot-labeled chloramphenicol antibody is present in kit with following solution form: the solution that the described quantum dot-labeled chloramphenicol antibody of every 25ug is obtained by the described diluted of 5ml.
Another object of the present invention is to provide a kind of method that detects chloromycetin in sample.
The method of chloromycetin in detection sample provided by the present invention, comprise 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, feed, honey or milk sample.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, by chloromycetin antigen direct coated in the micropore of polystyrene board, add the chloromycetin standard items or detect sample, and quantum dot-labeled chloramphenicol antibody, make it form Ag-Ab binary electrochemiluminescent immunoassay compound, excite and detect the fluorescence intensity of this immunofluorescence compound with fluorescence detector, by the concentration with measuring the typical curve contrast acquisition chloromycetin to be measured formed.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled chloramphenicol antibody, the chloromycetin of coated chloromycetin antigen in detecting sample is combined chloramphenicol antibody competitively, and the specific binding by Ag-Ab forms Ag-Ab binary immune complex.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled chloramphenicol antibody simultaneously and detecting sample, be coated on chloromycetin antigen in the polystyrene micropore chloromycetin in detecting sample and be combined with chloramphenicol antibody competitively, wherein with detect sample be combined remaining chloramphenicol antibody be coated on the chloromycetin antigen generation specific bond in microwell plate after form and be fixed on the Ag-Ab binary immune complex in microwell plate.
Chloromycetin immunofluorescence detection agent of the present invention is relevant to quantum dot-labeled immunofluorescence detection technique, to adopt quantum dot as the fluorescence signal marker material, carry out class methods of immunofluorescence quantitative measurement, this Technology Integration the research of the association areas such as the chemosynthesis of fluorescence quantum nano material, finishing and labelling technique, indirect competition formula immunoassay technology.
The present invention why can the chlorine detection mycin, be to adopt a kind of method of the immunofluorescence quantitative measurement based on quantum dot-labeled, be about to chloromycetin antigen direct coated in the micropore of polystyrene board, the measuring principle of the indirect competition immunofluorescence assay based on quantum dot-labeled, add the chloromycetin standard items or detecting sample, and after quantum dot-labeled chloramphenicol antibody, the fluorescence intensity that is attached to the Ag-Ab binary immune complex in the polystyrene board micropore by detection realizes the detection to chloromycetin: the quantity difference that is attached to the quantum dot-labeled antibody of polystyrene board micropore, the fluorescence intensity produced is also different.The content of the chloromycetin in the finite concentration scope in the height of fluorescence intensity level and sample is inversely proportional to.Can be made into typical curve by the chloromycetin standard items that add variable concentrations, the fluorescence intensity level that respectively detects sample according to this typical curve inquiry can obtain the concentration value of corresponding chloromycetin medicine.
Its concrete technical step comprises:
(1) preparation of fluorescence quantum point mark probe: adopt applicable water soluble fluorescence quantum dot, after activating its surperficial carboxyl, adopt the mode of chemical coupling that the chloramphenicol antibody orientation is connected to the quantum dot surface.
(2) envelope antigen: adopt chloromycetin antigen with the bovine serum albumin(BSA) coupling as envelope antigen, the method by physisorption by this antigen direct coated in the polystyrene board micropore.
(3) formation of Ag-Ab fluorescence immunoassay compound: add the chloromycetin standard items or detect sample in the above-mentioned polystyrene board micropore be coated with, and quantum dot-labeled chloramphenicol antibody, the chloromycetin antigen be adsorbed in hole combines with chloramphenicol antibody with the chloromycetin in standard or sample is emulative, by the specific binding formation Ag-Ab binary electrochemiluminescent immunoassay compound of Ag-Ab.
(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 the concentration with measuring the typical curve contrast acquisition chloromycetin to be measured formed.
The formation of described Ag-Ab binary electrochemiluminescent immunoassay compound is: after adding quantum dot-labeled chloramphenicol antibody, the coated chloromycetin of chloromycetin antigen in standard or sample is combined chloramphenicol antibody competitively, specific binding by Ag-Ab forms Ag-Ab binary immune complex, on it, the quantum dot of mark can fluoresce after exciting, 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 to excite and detect the fluorescence intensity of formed Ag-Ab binary electrochemiluminescent immunoassay compound with fluorescence microplate reader, because adopted antibody is fixed concentration, usually the concentration of the chloromycetin medicine in testing sample is higher, medication amount by antibody capture is more, the antibody of being combined with coated chloromycetin antigen is fewer, and the fluorescence intensity level recorded is lower.
Because quantum dot will carry out separation and purification with ultracentrifugation in antibody, carrying out coupling, the too little quantum dot of particle diameter can't be centrifugal as 8nm and 10nm's, can't purifying after coupling, and result of use is poor; The too large quantum dot of particle diameter is as the ratio more than 60nm is easier to assemble, and is used on kit homogeneity poor.
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) between 10～30%, preferably between 10～20%, preferably 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 quantum dot prepared by classic method is usually all lower than 40%, 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%.
For surveying for the residual chloromycetin quality testing, the quantum dot surface need be with the group be easy to the chloramphenicol antibody coupling, these groups can be carboxyl, amino groups, the group of optimizing is the surface functional group with carboxyl, usually adopt chemical method to connect antibody, with after EDC and NHS activation quantum dot, then close and react and complete coupling reaction with antibody generation carboxylic.
Before the condensate with the formation of peptide bond covalent bond by chloramphenicol antibody and quantum dot, also comprise the step that activates described quantum dot surface functional group.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 detected, and typically, high specificity, the antibody that affinity is high, can improve the accuracy of detection significantly.Research is found, is raising sensitivity, and described chloramphenicol antibody affinity costant is 10
6～10
8M
-1Described chloramphenicol antibody affinity costant is specially 10
7～10
8M
-1Described chloramphenicol antibody affinity costant especially is preferably 10
8M
-1.
Because chloromycetin is small-molecule substance, its molecular surface characteristic is unfavorable for the direct combination with polystyrene micropore plate, itself and carrier protein need to be carried out after coupling reaching by means of the character of surface of carrier protein the good combination with polystyrene micropore plate.Can be used as the seralbumin that various animals are arranged of carrier protein, as bovine serum albumin(BSA) (Bovine Serum Albumin, BSA), human serum albumins (Human Serum Albumin, HSA), also has keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH), the gamma globulin of thyroglobulin, albumin rabbit serum (RSA), ovalbumin (Ovalbumin, OVA), fibrinogen or rabbit and chicken.Research is found, the BSA physicochemical property is stable, lysine content is high, free amino group is many, larger solubleness is all arranged under different pH and ionic strength, all can carry out coupling with haptens in the situation that contain organic solvent (as pyridine, DMF etc.), and still keep solvable state after coupling, the splendid selection as carrier protein, therefore the present invention selects BSA as coupling protein.
The present invention is by the research to fluorescence quantum, chloromycetin antigen and chloramphenicol antibody molecular characterization, by the optimization to the preparation of various water soluble fluorescence quantum dot, coating and finishing condition, select applicable water soluble fluorescence quantum dot and specific antibody to carry out directed covalent chemical coupling, obtain functional fluorescence quantum point mark probe, and, by optimizing the various conditions of competitive immunization reaction, reach the quick and highly sensitive quantitative measurement to the residual chloromycetin medicine.Experiment showed, kit of the present invention highly sensitive, specificity good, accuracy is high.The inventive method can realize the quantitative detection to the residual chloromycetin 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 field of chloromycetin.
The accompanying drawing explanation
Fig. 1 water-soluble CdSe/ZnS fluorescence quantum Electronic Speculum (TEM) photo.
The typical curve of quantum dot-labeled indirect competition immuno-fluorescence assay chloromycetin for Fig. 2.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The composition of embodiment 1, detection kit and preparation
Chloromycetin opens a day Chemical Engineering Technology research institute purchased from the permanent unit in Beijing, and catalog number is 2857GBW (E) 060907.Its chemical name be D-Su Shi-(-)-N-[(-(hydroxymethyl)-(hydroxyl-p-nitrophenyl ethyl]-2, the 2-dichloro acetamide.
1, chloromycetin coating antigen
The conjugate of chloromycetin and carrier protein BSA, have hydroxyl on the chloromycetin molecule, can introduce accordingly carboxyl, then with the peptide bond that amino in BSA forms, be connected by the carboxyl in chloromycetin with BSA.
The synthetic employing diazotising method of CAP-BSA antigen, concrete steps are as follows:
1) get 50mg chloromycetin and be dissolved in the 1ml ethanol solution, with 1mol/L HCl, adjust PH to 1.0, then add 65 ℃ of reactions of 24mg zinc powder 40 minutes, obtain reactant liquor a;
2) reactant liquor a centrifugal (the centrifugal time is that 10min, centrifugal centrifugal force are 4000g, and centrifugal temperature is 4 ℃), get supernatant, with 1mol/L HCl, adjusts pH to 1.0, in 4 ℃, dropwise adds the NaNO that 180ul concentration is 1.45mol/L
2Aqueous solution, fully stirring reaction 3h, obtain solution b;
3) at 10ml 0.02mol/L, in the PBS that pH is 7.4, add 400mg BSA (Sigma-aldrich, 85041C) to be mixed with the BSA-PBS solution (solution c) of 40g/L,
4) by 1ml step 2) add BSA-PBS solution 10ml in gained solution b, be adjusted to pH to 8.5 with 2mol/l NaOH, carry out coupling reaction 6h under 4 ℃; Obtain reactant liquor d;
5) by reactant liquor d in 0.02mol/L, the 48h that dialyses in the PBS that pH is 7.4, every 12h changes a dislysate.Products obtained therefrom freeze dryer freeze-drying, in-20 ℃ of preservations, obtain chloromycetin antigen.
2, the chloromycetin coating antigen is coated
Adopting coated damping fluid is the coating buffer of concentration 10 μ g/ml by the dilution of chloromycetin coating antigen, and in 96 hole polystyrene micropore laths, every hole adds 100 μ l, in 4 ℃ of refrigerators, places and spends the night.Second day discards coating buffer, rinse each plate hole 3 times with cleansing solution after, each hole adds 200 μ l confining liquids, in 37 ℃ of sealings, processes 2h.After discarding afterwards confining liquid, with cleansing solution, rinsing each plate hole 3 times, carry out vacuum drain, with after the aluminium foil bag sealing, being positioned over-20 ℃ of preservations.
3, quantum dot-labeled chloramphenicol antibody:
Chloramphenicol antibody is 10 for tiring
6, affinity costant is 10
8M
-1The chloromycetin monoclonal antibody, it is purchased from Yunnan University's monoclonal antibody engineering center, catalog number is CAP-001.
Quantum dot is purchased from Shenzhen TELUS Science and Technology Ltd., and catalog number is
LumiQD
TM20.The sign of this quantum dot is as follows: the CV that particle diameter is 20nm, particle diameter is 15%, and quantum yield is 60%, and surperficial carboxyl-content is 5 * 10
-3Mmol/mg, water-soluble, the CdSe/ZnS nucleocapsid structure, excitation wavelength is 345nm, emission wavelength is 620nm; The red fluorescence quantum dot.The scintigram of quantum dot as shown in Figure 2.
In described quantum dot-labeled chloramphenicol antibody, the amino on the carboxyl on described quantum dot and described chloramphenicol antibody forms peptide bond, and then described quantum dot is connected with described chloramphenicol antibody.
Quantum dot marking method is:
1) get the MES damping fluid of 0.1M for the above-mentioned quantum dot of 2.5mg (take 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water, adjust pH to 4.7) wash and use the 20000rpm centrifugal enrichment to remove supernatant after, be that 0.1M, pH value are 4.7 the MES damping fluid is resuspended by 1ml concentration, add the 1-ethyl of 0.96mg (final concentration is 5mM)-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15mg (final concentration is 10mM) N-maloyl imines (NHS) in wherein.Temperature of reaction is 37 ℃, after reacting half an hour, and the quantum dot after being activated;
2), with the borate buffer solution washing of 50mM pH=8.5, get the borate buffer solution that quantum dot after 0.15mg chloromycetin monoclonal antibody and 2.5mg activation is mixed into 0.8ml 50mM pH=8.5 and (take 1.9g Na
2B
4O
7.10H
2O is dissolved in the 100ml pure water, adjusts pH to 8.5) in fully mix.The lower reaction of room temperature (25 ℃) 3.5 hours, allow antibody and quantum dot form stable peptide bond covalent bond, obtains containing the reactant liquor of quantum dot after coupling;
3) after reaction finishes, to step 2) add the BSA (Sigma-Aldrich that final concentration is 5% (quality percentage composition) in the reactant liquor that obtains, 85041C) the residual activity amino sites is sealed, reaction is carried out 0.5 hour under 37 ℃, obtains the reactant liquor that contains the rear quantum dot of sealing; After completing, with the 0.02M PBS damping fluid of pH=7.4, (take 2.3g Na
2HPO
4, 0.524g NaH
2PO
4.H
2O, 8.77g NaCL are dissolved in the 1L pure water, adjust pH to 7.4) washing, the 20000rpm centrifugal enrichment is removed supernatant, and resuspended rear 4 ℃ of preservations are stand-by, obtain quantum dot-labeled chloramphenicol antibody.
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L 4, chloromycetin standard items: with dilution, chloromycetin is diluted to the solution of following each concentration:;
5, the PBS damping fluid of dilution: 0.02M, pH7.4;
Preparation: take 2.3g Na
2HPO
4, 0.524g NaH
2PO
4.H
2O and 8.77g NaCL are dissolved in the 1L pure water, adjust pH to 7.4.
6, cleansing solution: PBST damping fluid; Get the NaN of 0.2ml Tween20 and 0.1g
3Be dissolved in above-mentioned PBS damping fluid, after dissolving, with above-mentioned PBS damping fluid, be settled to 1L.
7, the carbonate buffer solution of coated damping fluid: 0.05M, pH9.6.
8, confining liquid: 10g BSA and 0.2ml Tween20 are dissolved in above-mentioned PBS damping fluid, after dissolving, with the PBS damping fluid, are settled to 1L.
The preparation method of embodiment 2, typical curve
Add in the chloromycetin micropore lath prepared that concentration is 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5, the chloromycetin standard solution of 10ug/L, the 50ul/ hole, by PBS-T dilution dilution [being the 25ug labelled antibody: the 5ml dilution] in 1: 50 for quantum dot-labeled ENR antibody, add 50ul in each micropore, room temperature vibration 1 hour, detect 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.
When the detectability that competition is detected is decided to be B0/B=1.2, 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), determine the sensitivity of detection system according to Regression Equations.Testing result is as shown in table 1 below, and detection limit is decided to be to 0.001ug/L.
The quantum dot kit detected value of table 1 chloromycetin variable concentrations sample
The mensuration of embodiment 3, cross reaction
Select chloramphenicol sodium succinate (Sigma-aldrich, C3787-5G), (a day Chemical Engineering Technology research institute is opened to Florfenicol by the permanent unit in Beijing, 30242CDCT-C13665000), (a day Chemical Engineering Technology research institute is opened to Thiamphenicol by the permanent unit in Beijing, 14814NIC-130433), ammonia Bian XiLin (Sigma-aldrich, A9393-5G) and sulfamethazine (a day Chemical Engineering Technology research institute is opened by the permanent unit in Beijing, 30137CDCT-C16996500) 5 kinds of medicines, be made into respectively series concentration, detected with quantum dot kit.Calculate the IC50 that respectively competes thing, calculate respectively the cross reacting rate of these 5 kinds of medicines and chloromycetin quantum dot kit with following formula.Computing formula is: cross reacting rate (%)=[IC50 (chloromycetin)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.Result shows the chloromycetin quantum dot kit except chloramphenicol sodium succinate is had higher cross reaction and all the other 4 kinds of cross reacting rates all are less than to 0.1%.
The cross reaction of table 2 chloromycetin quantum dot kit and other medicines
The mensuration of embodiment 4, accuracy
(1) sample extraction:
1, tissue sample: the meat sample is used homogenate after removing adipose tissue; The direct homogenate of peeled shrimp sample; The casing sample is cut into clean scissors the fragment that length is no more than 5mm, when the casing salinity is more, first uses rinsed with deionized water 20min with desalination, after draining, shreds again.Take sample 5g (being accurate to 0.01g) to centrifuge tube, add 15mL acetonitrile-aqueous solution (84+16), vortex vibration 1min, the centrifugal 15min of 4000g.Pipette the 3ml supernatant to glass test tube, add 3ml ethyl acetate and 1ml 0.5mol/L sodium chloride solution, vibration 1min, remove lower floor's water after stratification, organic phase dries up with nitrogen, and residue dissolves residue with 1ml 0.02M PBS, add 1ml normal hexane vibration 1min, the centrifugal 10min of 2000g, after removing the normal hexane and fat deposit on upper strata, get 50ul lower floor solution for detection of.
2, urine sample: the urine of clarification can be directly used in detection, if cloudy urine needs first centrifugal (4000g) 10min, gets supernatant and is detected.
3, feed: take feed 3g (being accurate to 0.01g), add ethyl acetate 6ml, fully mix, vibration 10min, the centrifugal 10min of 4000g, get upper organic phase 4ml (sample that approximately is equivalent to 2g), and 50 ℃ of lower nitrogen dry up, residue dissolves residue with 1ml 0.02M PBS, get 50ul for detection of.
4, honey: the sample to without crystallization stirs it; To the sample of crystallization is arranged, under encapsulation situations, be placed in the water-bath that is no more than 60 ℃ warm, vibration stirs evenly after sample all melts, and is cooled to room temperature.Take 2g sample (being accurate to 0.01g), be placed in 25m L tool plug centrifuge tube, add 4ml water and 4ml ethyl acetate, fully mix, sample is dissolved fully.The 10min that vibrates on oscillator, with the centrifugal 10min of 4000g.Accurately draw upper strata ethyl acetate 2ml in 10ml tool plug test tube, dry up with nitrogen under 50 ℃.Add 1ml 0.02M PBS buffer solution to dissolve residue, get 50ul for detection of.
5, milk sample: the 2ml that materialses, add ethyl acetate 4ml, vibration 10min, the centrifugal 10min of room temperature 4000g, pipette ethyl acetate 2ml supernatant liquid (being equivalent to the 1ml milk sample), and 50 ℃ of lower nitrogen dry up.Add 0.5ml0.02M PBS buffer solution to dissolve residue, get 50ul for detection of.
(2) mensuration of the recovery
Detect 20 parts of negative pig urine samples, wherein 5 parts of negative urine samples are added the CAP 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 the recovery that chloromycetin adds sample is 83%～109%, average recovery rate 96.4%, and the coefficient of variation 6.02%～10.09%, average coefficient of variation 7.602%, accuracy is better.
Table 3 determination of recovery rates
Claims (4)
1. the box of the immunofluorescence detection agent for detection of chloromycetin, comprise chloromycetin coating antigen and quantum dot-labeled chloramphenicol antibody;
Described chloramphenicol antibody is that antibody titer is 10
6Above, antibody affinity costant is 10
6～10
8M
-1The chloromycetin 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 core shell structure; The quantum yield of described quantum dot is 40～70%; The excitation wavelength of described quantum dot is 345nm, and emission wavelength is 620nm;
The particle diameter of described quantum dot is 10～20nm, and the deviation of its particle diameter is between 10～30%;
The conjugate that described chloromycetin coating antigen is chloromycetin and carrier protein;
Also comprise chloromycetin standard items, dilution, cleansing solution in described kit, contain porose polystyrene board, coated damping fluid and confining liquid;
Described chloromycetin standard items be D-Su Shi-(-)-N-[(-(hydroxymethyl)-(hydroxyl-p-nitrophenyl ethyl]-2, the 2-dichloro acetamide;
Described dilution is the PBS damping fluid;
Described cleansing solution is the PBST damping fluid;
Described coated damping fluid is carbonate buffer solution;
Described confining liquid is the PBS damping fluid containing being useful on coated albumen;
The standard items that described standard items are following solution form: with described dilution by described D-Su Shi-(-)-N-[(-(hydroxymethyl)-(hydroxyl-p-nitrophenyl ethyl]-2, the 2-dichloro acetamide; 0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L be diluted to the solution of following each concentration:.
2. kit according to claim 1, it is characterized in that: described chloromycetin coating antigen is coated on described containing on porose polystyrene board, method for coating, for described coated damping fluid, to dilute the coating buffer that described chloromycetin coating antigen obtains 10 μ g/ml, adds 100 μ l in every hole.
3. kit according to claim 2, it is characterized in that: described quantum dot-labeled chloramphenicol antibody is present in kit with following solution form: the solution that the described quantum dot-labeled chloramphenicol antibody of every 25ug is obtained by the described diluted of 5ml.
4. a method that detects chloromycetin in sample, comprise the steps: with arbitrary described kit in claim 1-3, testing sample to be detected, and described testing sample is animal muscle tissue's sample, animal urine, feed, honey or milk sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101893077A CN102288765B (en) | 2011-07-07 | 2011-07-07 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
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| CN103389377A (en) * | 2012-05-08 | 2013-11-13 | 上海大学 | Liquid-phase chip method for detecting chloromycetin in aquatic product |
| CN103389378A (en) * | 2012-05-08 | 2013-11-13 | 上海大学 | Kit used for detecting chloromycetin residue in aquatic product |
| CN108872169B (en) * | 2018-05-07 | 2021-02-05 | 广西大学 | Method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue |
| CN108956572B (en) * | 2018-07-28 | 2023-05-12 | 刘振平 | Method for detecting chloramphenicol in honey based on manganese-doped zinc sulfide quantum dot phosphorescence signals |
| CN109781691B (en) * | 2019-02-28 | 2021-11-30 | 渤海大学 | Method for detecting chloramphenicol based on polystyrene nano fluorescent microsphere probe |
| CN109917123B (en) * | 2019-04-19 | 2024-02-13 | 广州安诺科技股份有限公司 | Pesticide residue detection device and detection method based on DELFIA |
| CN110845619B (en) * | 2019-09-04 | 2021-08-03 | 山东绿都生物科技有限公司 | Monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof |
| CN112946040B (en) * | 2021-02-04 | 2023-01-17 | 苏州大学 | Electrochemical luminescence immunosensor and application thereof in florfenicol detection |
| CN114015434B (en) * | 2021-10-18 | 2024-01-23 | 中国科学院化学研究所 | Perovskite nanocrystalline kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1945331A (en) * | 2006-10-20 | 2007-04-11 | 邹明强 | Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds |
| CN101308146A (en) * | 2008-06-17 | 2008-11-19 | 江南大学 | Method for quantum dot mark indirect competition fluoroimmunoassay detection for becort |
| CN201438191U (en) * | 2009-04-02 | 2010-04-14 | 北京望尔康泰生物技术有限公司 | Chloromycetin ELISA detection kit |
| CN101762706A (en) * | 2010-01-05 | 2010-06-30 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
| CN101907625A (en) * | 2010-07-08 | 2010-12-08 | 浙江大学 | Method for preparing quantum dot immune fluorescent probe |
| CN101915840A (en) * | 2010-07-20 | 2010-12-15 | 浙江大学 | Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody |
| CN101942414A (en) * | 2010-01-21 | 2011-01-12 | 泰州康正生物技术有限公司 | Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008116044A1 (en) * | 2007-03-20 | 2008-09-25 | Emory University | Semiconductor quantum dots for efeicient delivery and intracellular imaging of sirna |
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2011
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1945331A (en) * | 2006-10-20 | 2007-04-11 | 邹明强 | Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds |
| CN101308146A (en) * | 2008-06-17 | 2008-11-19 | 江南大学 | Method for quantum dot mark indirect competition fluoroimmunoassay detection for becort |
| CN201438191U (en) * | 2009-04-02 | 2010-04-14 | 北京望尔康泰生物技术有限公司 | Chloromycetin ELISA detection kit |
| CN101762706A (en) * | 2010-01-05 | 2010-06-30 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
| CN101942414A (en) * | 2010-01-21 | 2011-01-12 | 泰州康正生物技术有限公司 | Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same |
| CN101907625A (en) * | 2010-07-08 | 2010-12-08 | 浙江大学 | Method for preparing quantum dot immune fluorescent probe |
| CN101915840A (en) * | 2010-07-20 | 2010-12-15 | 浙江大学 | Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody |
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