CN101162230A - Kit for quantitative determining enrofloxacin content in food product and testing method thereof - Google Patents
Kit for quantitative determining enrofloxacin content in food product and testing method thereof Download PDFInfo
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- CN101162230A CN101162230A CNA2007101502838A CN200710150283A CN101162230A CN 101162230 A CN101162230 A CN 101162230A CN A2007101502838 A CNA2007101502838 A CN A2007101502838A CN 200710150283 A CN200710150283 A CN 200710150283A CN 101162230 A CN101162230 A CN 101162230A
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Abstract
The invention relates to a reagent kit for quantificationally detecting the content of enrofloxacin in foods, belonging to the immunoassay technical field, consisting of a covering plate and a reagent group, wherein, the reagent group comprises buffer solution (1), a zymolyte A (2), a zymolyte B (3), scrub solution (4), stop solution (5), enrofloxacin standard solution (6) and enzyme-marked enrofloxacin antigen solution (7); a millipore of the covering plate is covered with polyclone enrofloxacin antibody. The invention has the characteristics of simple manipulation, high detection sensitivity, strong specificity, good accuracy and low cost of detection. Moreover, the invention is of wide range of application. With economic and social benefits, the invention is a reagent kit for quantificationally detecting the content of enrofloxacin in foods with high creativity and good potential applicaiton.
Description
Technical field
The invention belongs to the immuno analytical method field, the kit and the detection method thereof of determining enrofloxacin content in especially a kind of detection by quantitative food.
Background technology
The bright again ethyl Ciprofloxacin of Enrofloxacin (Enrofloxacin), chemical constitution is (C
19H
22FN
3O
3), molecular weight is 359.1646.Enrofloxacin can cause toxic and side effects such as neurotoxic, renal dysfunction, allergic reaction, cub arthritis, its residual human body health that directly threatens in animal food.It is residual except the direct harm of its toxic and side effect to the mankind, the medicine of even more serious is residual low concentration in the animal food induces the human disease bacterium to produce drug resistance easily, thereby be unfavorable for of the treatment of such medicine, therefore must attach great importance to the residue problem of Enrofloxacin in animal food human diseases.Enrofloxacin and metabolic product Ciprofloxacin thereof are more and more at bad reaction and drug resistance report that animal doctor and people doctor is produced when using clinically in recent years.Because the drug resistance of Enrofloxacin and potential carcinogenicity, reaching the maximum residue limit(MRL) that China all formulated in tissue abroad is 100 μ g/kg.U.S. FDA regulation Enrofloxacin is forbidden in food animal; European Union (E C) regulation Enrofloxacin maximum residue limit(MRL) (MRL) in the liver of ox, pig, poultry, kidney, muscle is 30 μ g/kg; The Enrofloxacin MRL that The World Health Organization (WHO) is recommended is 40 μ g/kg; Area, Hong Kong regulation Enrofloxacin MRL is 100 μ g/kg; The MRL of China's regulation Enrofloxacin in egg, milk is 100 μ g/kg.
Detection method to Enrofloxacin mainly comprises at present: the microorganism as prescreening method suppresses experiment and immunization method, thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry method (GC/MS), fluorescence spectrophotometry, euzymelinked immunosorbent assay (ELISA) (ELISA).Wherein, what research both at home and abroad and application were more is the HPLC method, but this method pre-treatment process is loaded down with trivial details, complicated operation, and need the professional to operate usually, the instrument and equipment costliness, testing process is consuming time and expense is higher, is unsuitable for the field quick detection of a large amount of samples.Enzymoimmunoassay ELISA is owing to its high specificity, and highly sensitive, accuracy is good, and is easy and simple to handle, and the advantages such as detection that are suitable for batch samples more and more are subjected to people's favor.Because the technical conditions of ELISA require low, easy and simple to handle, easy commercialization, economical and practical, are the method for quick of food veterinary drug residue first-selection.This problem is intended research and is set up the Enrofloxacin enzyme-linked immune detection method, and this is to improving China's food inspection system, and it is significant to ensure food safety.
Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) ultimate principle is: enzyme and antigen or antibody are combined with crosslinking chemical, corresponding antibodies or antigen generation idiosyncrasy in this kind enzyme-labelled antigen or antibody and the testing sample, and strong bonded, when adding the substrate of corresponding enzyme, substrate is generated the colour generation product by enzymatic, can do qualitative or quantitative observation with the colour generation depth according to having or not of colour generation thing.Because this technology is to be based upon on the basis of efficient catalytic effect of antigen-antibody reaction and enzyme, so this technology has characteristics such as detection sensitivity height, high specificity, accuracy are good.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of simple to operate, detection sensitivity is high, high specificity, accuracy are good, detect the kit and the detection method thereof of determining enrofloxacin content in the low detection by quantitative food of cost.
The present invention is achieved through the following technical solutions:
The kit of determining enrofloxacin content in a kind of detection by quantitative food, kit is made of by plate reagent set and bag, wherein:
(1). reagent set
1.. damping fluid: be phosphate buffer, pH7.4 dilutes by 1: 5 with secondary water during use;
2.. substrate A: for containing the sodium-acetate buffer of carbamide peroxide;
3.. substrate B: for contain 3,3 ', 5,5 '-dimethyl sulphoxide solution of tetramethyl benzidine TMB;
4.. cleansing solution: for containing the phosphate buffer of 2.5% tween; Dilute according to 1: 5 with secondary water during use;
5.. stop buffer: be 1.25mol/L sulfuric acid;
6.. the Enrofloxacin titer: its Enrofloxacin concentration of standard solution is 100 μ g/L, with after the damping fluid dilution, presses six gradients of dilution in 1: 4 again during use;
7.. enzyme labeling Enrofloxacin antigenic solution: be Enrofloxacin-horseradish peroxidase connector ENF-HRP, use damping fluid during use according to ten thousand times of dilutions in 1: 20;
(2). bag is by plate
Bag is coated with polyclone Enrofloxacin antibody in the micropore of plate, and adopts Na
2CO
3-NaHCO
3Damping fluid with this polyclone Enrofloxacin antibody dilution as coating buffer, each hole of micropore adds coating buffer in wrapping by plate, place in room temperature and to spend the night or 35～40 ℃ of constant temperature incubations 2～3 hours, the cleansing solution washing is removed coating buffer three times, adding the freeze-drying fluid-tight closed 0.8～1.2 hour, after the cleansing solution washing four times, freeze-drying, 3～5 ℃ of preservations.
And described bag is 96 holes or 48 hole microwell plates by plate, described damping fluid Na
2CO
3-NaHCO
3The pH value be 9.6.
And, described bag is to select for use female White Rabbit to carry out five immunity of artificial antigen ENF-KLH by the polyclone Enrofloxacin antibody in the plate micropore, back 10 days of three immunity are gathered centrifugal blood by the auricular vein of rabbit and are obtained the antiserum detection of tiring, adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the Sodium azide of gained antibody interpolation 0.1% (W/V).
And, described artificial antigen ENF-KLH is with Enrofloxacin, N-hydroxy-succinamide and N, the N-dicyclohexylcarbodiimide is dissolved in N, in the dinethylformamide, 360 ° of room temperatures of aluminium film parcel were shaken 4～6 hours, the centrifugal precipitation of removing slowly joins the upper strata activated ester solution in the sodium bicarbonate solution that is dissolved with KLH under condition of ice bath, 360 ° of reactant liquors is shaken up reaction under 4 ℃ and spends the night, the bag filter of then reactant liquor being packed into, dialyse in 4 ℃ of following phosphate buffers, accurately measure the volume of protein conjugate solution then, measure concentration and add Sodium azide, packing ,-20 ℃ of preservations.
And described bag is the phosphate buffer that contains skimmed milk power by the freeze-drying liquid in the plate.
And described connector ENF-HRP is with Enrofloxacin, N-hydroxy-succinamide, N, and N '-dicyclohexylcarbodiimide is dissolved in N, and in the dinethylformamide, 360 ° of room temperatures of aluminium film parcel were shaken the centrifuging and taking supernatant 4～6 hours; Horseradish peroxidase HRP is dissolved in the sodium bicarbonate solution, under ice bath, be added dropwise to Enrofloxacin haptens reactant liquor in the HRP solution, under dripping fully back 4 ℃ 360 ° of reactant liquors are shaken up reaction and spend the night, at last all reactant liquors are put into the phosphate buffer dialysis 72h of bag filter with 0.01mol/L.
The detection method of the kit of determining enrofloxacin content in a kind of detection by quantitative food, its detection method may further comprise the steps:
(1). when temperature is 10～30 ℃, carry out sample pre-treatments, obtain sample extracting solution;
(2). open kit and take out bag by plate, Enrofloxacin titer and sample extracting solution with various concentration joins bag by in the plate micropore separately respectively, and the parallel application of sample of diplopore, and then in various concentration Enrofloxacin titers and sample extracting solution micropore separately, add enzyme-labelled antigen solution, concussion mixing 2～8min, room temperature reaction 0.8～1.5 hour;
(3)., by plate 3～4 times liquid in the hole is got rid of with cleansing solution wash bags, buckle with thieving paper and do, add the mixed liquor of substrate A and substrate B, reacted under the room temperature 0.4～0.6 hour;
(4). in each micropore, add stop buffer, cessation reaction, read absorbance with microplate reader, draw the canonical plotting of Enrofloxacin according to the absorbance of various concentration Enrofloxacin titers, the canonical plotting of contrast Enrofloxacin obtains contents of enrofloxacin in the sample extracting solution.
And the proportioning of the mixed liquor of described substrate A and substrate B is 14.6: 0.45.
And sample thief adds extract, and oscillator mixing 2min is centrifugal, gets the part supernatant and dilutes with phosphate buffer.
And described extract is an acetonitrile: 0.1M NaOH is 4: 1 mixed solution by volume.
Beneficial effect of the present invention and advantage are:
1. the present invention has simple to operate, characteristics such as detection sensitivity is high, high specificity, accuracy is good, the detection cost is low, and the scope of application is wider.Mainly be applicable to the detection by quantitative of the determining enrofloxacin content in food and the meat.Its antibody has good broad spectrum activity and sensitivity, and gained antibody and enzyme-labelled antigen are stable, has long advantage of preservation under room temperature time.
2. method for quick provided by the invention is easy and simple to handle, quick, finish whole detecting operation processes and only need several hours, and the degree of accuracy that detects can reach more than 90%, and detection sensitivity can reach μ g/kg level and be fit to very much the on-the-spot needs that detect.Because antibody and enzyme-labelled antigen, at normal temperatures can 1 week of preservation, the ELISA Plate that is equipped with of enzyme-labelled antigen bag can preservation 4 ℃ under more than 6 months, thus be the centralized detecting of carrying out extensive sample, great convenience is provided.
3. the present invention is with low cost, detection efficiency is high, easy and simple to handle, not only have an economic benefit but also social benefit is arranged, be determining enrofloxacin content kit and detection method thereof in the higher detection by quantitative food that has a good application prospect of a kind of novelty.
Description of drawings
Fig. 1 Enrofloxacin of the present invention-ELISA canonical plotting.
Embodiment
The present invention is described in further detail by following examples, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The present invention is the kit of determining enrofloxacin content in the detection by quantitative food, is made of by plate and reagent set bag, wherein consisting of of this reagent set:
(1) damping fluid: be phosphate buffer, pH7.4 dilutes by 1: 5 with secondary water during use;
(2) substrate A: for containing the sodium-acetate buffer of carbamide peroxide;
(3) substrate B: for contain 3,3 ', 5,5 '-dimethyl sulphoxide solution of tetramethyl benzidine TMB;
(4) cleansing solution: for containing the phosphate buffer of 2.5% tween; Dilute according to 1: 5 with secondary water during use;
(5) stop buffer: be 1.25mol/L sulfuric acid;
(6) Enrofloxacin titer: its Enrofloxacin concentration of standard solution is 100 μ g/L, after damping fluid is diluted to 10 μ g/L during use, again by six gradients of dilution in 1: 4;
(7) enzyme labeling Enrofloxacin antigenic solution: be Enrofloxacin-horseradish peroxidase connector (ENF-HRP), use damping fluid during use according to ten thousand times of dilutions in 1: 20.
The detection method of the kit of determining enrofloxacin content in a kind of detection by quantitative food: may further comprise the steps:
(1). when temperature is 10～30 ℃, carry out sample pre-treatments, obtain sample extracting solution;
(2). open kit and take out bag by plate, Enrofloxacin titer and sample extracting solution with various variable concentrations joins bag by in the plate micropore separately respectively, and the parallel application of sample of diplopore, and then in various variable concentrations Enrofloxacin titers and sample extracting solution micropore separately, add enzyme-labelled antigen solution, concussion mixing 5min, room temperature reaction 0.8～1.5 hour;
(3)., by plate 3～4 times liquid in the hole is got rid of with cleansing solution wash bags, buckle with thieving paper and do, add the mixed liquor of substrate A and substrate B, reacted under the room temperature 0.4～0.6 hour;
(4). in each micropore, add stop buffer, cessation reaction, read absorbance with microplate reader, draw the canonical plotting of Enrofloxacin according to the absorbance of various variable concentrations Enrofloxacin titers, the canonical plotting of contrast Enrofloxacin obtains contents of enrofloxacin in the sample extracting solution.
Below by an instantiation, effect of the present invention is narrated.
Detect contents of enrofloxacin in the chicken meat sample:
(1). sample pre-treatments: get each 4g of chicken meat sample respectively, adding 10mL extract (acetonitrile: 0.1M NaOH by volume 4: 1), oscillator mixing 2 minutes, after the extraction through centrifugal (15 ℃, 3500g, 10 minutes), get the 20 times of dilutions of phosphate buffer of part supernatant;
(2). open kit and take out bag by plate, titer with 100 μ L Enrofloxacin gradient dilutions joins bag by in the plate micropore separately with the parallel application of sample of 100 μ L sample extracting solution diplopores respectively, and then in Enrofloxacin titer and sample extracting solution micropore separately, respectively add the enzyme-labelled antigen solution of 100 μ L, ten thousand times of dilutions in 1: 20, shook room temperature reaction 1h even 5 minutes;
(3). wash plate 3 times with cleansing solution; Liquid in the hole is got rid of, buckle with thieving paper and do, add the mixed liquor of 150 μ L substrate A and substrate B in every hole, reaction is 0.5 hour under the room temperature;
(4). in sample extracting solution and Enrofloxacin titer micropore separately, add 50 μ L stop buffers, carry out cessation reaction, under dual wavelength mode (450-650nm), read absorbance with microplate reader, draw the canonical plotting of Enrofloxacin, the canonical plotting of contrast Enrofloxacin obtains contents of enrofloxacin in the sample extracting solution.
(5) result's calculating
1) calculates the inhibiting rate value
By formula (A) calculates the inhibiting rate of variable concentrations Enrofloxacin to antigen-antibody binding reaction:
In the formula:
IC%---Enrofloxacin is to the inhibiting rate of antigen-antibody binding reaction;
The difference of A---450nm light absorption value and 650nm light absorption value;
The mean light absorbency value of A sample---Enrofloxacin titer or sample liquid;
The A blank---do not add the mean light absorbency value of enzyme mark and Enrofloxacin titer.
2) drawing standard curve
With the inhibiting rate is ordinate, and Enrofloxacin concentration logarithm is that horizontal ordinate is drawn calibration curve.Each test all should repaint typical curve, sees Fig. 1.
3) result calculates
The typical curve of drawing from Fig. 1 reads the pairing Enrofloxacin concentration of sample liquid inhibiting rate (C), and by formula (B) calculates the enrofloxacin residual amount in the sample:
X＝C×R....................................(B)
In the formula:
X---enrofloxacin residual amount in the sample, unit is every kilogram of microgram (μ g/kg);
C---the inhibiting rate in hole checks in Enrofloxacin concentration in the sample per sample, and unit is every liter of microgram (μ g/L);
R---reduction coefficient, for example chicken is 50.
The mark-on test:
In the mark-on test, in sample, add the Enrofloxacin of variable concentrations respectively, with the abundant mixing of mark-on sample, room temperature standing over night.All the other processing procedures are the same.In detection to chicken meat sample, carry out the mark-on recovery test in 10,20 and 40 μ g/kg levels respectively, its recovery is 82% and 72.5% and 79.25%.
The preparation of synthetic Enrofloxacin antigenic solution:
Accurately take by weighing Enrofloxacin 0.02～0.03mmol respectively, 0.02～0.04mmol N-hydroxy-succinamide and 0.05～0.07mmol N, the N-dicyclohexylcarbodiimide is dissolved in 150 μ L N, in the dinethylformamide, 360 ° of room temperatures of aluminium film parcel were shaken 4-6 hour, centrifugal (5 ℃, 10000g, 5 minutes) remove precipitation, the upper strata activated ester solution slowly being joined 3mL under condition of ice bath is dissolved with the sodium bicarbonate of 10mgKLH (130mmol/L pH=8.1) in the solution, shakes up reaction with 360 ° of reactant liquors under 4 ℃ and spends the night, the bag filter of then reactant liquor being packed into, under 4 ℃, dialyse in the phosphate buffer of the 0.01mol/mL of pH=7.4, accurately measure the volume of protein conjugate solution then, measure concentration and add 0.1% (W/V) Sodium azide, packing ,-20 ℃ of preservations.
The preparation of enzyme labeling Enrofloxacin antigenic solution:
Accurately take by weighing Enrofloxacin haptens 0.02～0.03mmol respectively and be dissolved in 150 μ L N, in the dinethylformamide (DMF), add 0.02～0.04mmol N-hydroxy-succinamide (NHS) and 0.05～0.07mmol N, N-dicyclohexylcarbodiimide (DCC), 360 ° of room temperatures of aluminium film parcel were shaken 4-6 hour, there is dregs to produce, centrifugal (5 ℃, 10000g, 5 minutes) remove precipitation, under condition of ice bath, the upper strata activated ester solution slowly joined the sodium bicarbonate (130mmol/L that 3mL is dissolved with 10mgHRP, pH=8.1) in the solution, be put in 4 ℃ of following stirring reactions and spend the night, the bag filter of then reactant liquor being packed into, under 4 ℃, dialyse in the phosphate buffer of the 0.01mol/mL of pH=7.4, accurately measure the volume of protein conjugate solution then, measure concentration and add thimerosal, 4 ℃ of preservations
Bag is by the preparation of plate:
Polyclone Enrofloxacin antibody sandwich is used pH9.6Na in microwell plate
2CO
3-NaHCO
3Damping fluid be that 1 μ g/100 μ L is as coating buffer with polyclone Enrofloxacin antibody dilution, the every hole of 96 hole microwell plates adds 100 μ L, room temperature is placed and to be spent the night or 37 ℃ of constant temperature incubations 2～3 hours, the cleansing solution washing is removed coating buffer three times, adding 200 μ L freeze-drying fluid-tights closed 1 hour, after the cleansing solution washing four times, freeze-drying, 4 ℃ of preservations.
Enrofloxacin is a kind of micromolecule haptens, and molecular weight is 359.16, has only reactionogenicity and non-immunogenicity, need with carrier protein couplet after just can make animal produce immune response.This test is carried out animal immune with synthetic Enrofloxacin-kLH as immunogene.
The preparation of polyclone Enrofloxacin antibody:
1, immune animal is selected 3 of Japan large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are numbered respectively No. 1, No. 2 and No. 3.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
2, immune programme for children: initial immunity: for improving the immunity of antigen, adopt the immunogene by Freund's complete adjuvant emulsification, dosage is 1mg (being dissolved in the NaCl of 1mL 0.9% and the Freund's complete adjuvant of 1mL).Booster immunization: carried out booster immunization on the 14th day behind the initial immunity, dosage is 0.5mg (being dissolved in the NaCl of 1mL 0.9% and the incomplete Freund of 1mL).Later on once, be total to immunity 6 times every 30 days booster immunizations.Since the 2nd booster immunization, each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and affinity is measured.Adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the Sodium azide of gained antibody interpolation 0.1% (W/V).
Claims (10)
1. the kit of determining enrofloxacin content in the detection by quantitative food, kit is made of by plate reagent set and bag, it is characterized in that:
(1). reagent set
1.. damping fluid: be phosphate buffer, pH7.4 dilutes by 1: 5 with secondary water during use;
2.. substrate A: for containing the sodium-acetate buffer of carbamide peroxide;
3.. substrate B: for contain 3,3 ', 5,5 '-dimethyl sulphoxide solution of tetramethyl benzidine TMB;
4.. cleansing solution: for containing the phosphate buffer of 2.5% tween; Dilute according to 1: 5 with secondary water during use;
5.. stop buffer: be 1.25mol/L sulfuric acid;
6.. the Enrofloxacin titer: its Enrofloxacin concentration of standard solution is 100 μ g/L, with after the damping fluid dilution, presses six gradients of dilution in 1: 4 again during use;
7.. enzyme labeling Enrofloxacin antigenic solution: be Enrofloxacin-horseradish peroxidase connector ENF-HRP, use damping fluid during use according to ten thousand times of dilutions in 1: 20;
(2). bag is by plate
Bag is coated with polyclone Enrofloxacin antibody in the micropore of plate, and adopts Na
2CO
3-NaHCO
3Damping fluid with this polyclone Enrofloxacin antibody dilution as coating buffer, each hole of micropore adds coating buffer in wrapping by plate, place in room temperature and to spend the night or 35～40 ℃ of constant temperature incubations 2～3 hours, the cleansing solution washing is removed coating buffer three times, adding the freeze-drying fluid-tight closed 0.8～1.2 hour, after the cleansing solution washing four times, freeze-drying, 3～5 ℃ of preservations.
2. the kit of determining enrofloxacin content in the detection by quantitative food according to claim 1 is characterized in that: described bag is 96 holes or 48 hole microwell plates by plate, described damping fluid Na
2CO
3-NaHCO
3The pH value be 9.6.
3. the kit of determining enrofloxacin content in the detection by quantitative food according to claim 1, it is characterized in that: described bag is to select for use female White Rabbit to carry out five immunity of artificial antigen ENF-KLH by the polyclone Enrofloxacin antibody in the plate micropore, back 10 days of three immunity are gathered centrifugal blood by the auricular vein of rabbit and are obtained the antiserum detection of tiring, adopt the arteria carotis blood collection method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the Sodium azide of gained antibody interpolation 0.1% (W/V).
4. the kit of determining enrofloxacin content in the detection by quantitative food according to claim 3, it is characterized in that: described artificial antigen ENF-KLH is with Enrofloxacin, N-hydroxy-succinamide and N, the N-dicyclohexylcarbodiimide is dissolved in N, in the dinethylformamide, 360 ° of room temperatures of aluminium film parcel were shaken 4～6 hours, the centrifugal precipitation of removing, under condition of ice bath, the upper strata activated ester solution is slowly joined in the sodium bicarbonate solution that is dissolved with KLH, under 4 ℃ 360 ° of reactant liquors being shaken up reaction spends the night, the bag filter of then reactant liquor being packed into, dialyse in 4 ℃ of following phosphate buffers, accurately measure the volume of protein conjugate solution then, measure concentration and add Sodium azide, packing ,-20 ℃ of preservations.
5. determining enrofloxacin content kit in the detection by quantitative food according to claim 1 is characterized in that: described bag is the phosphate buffer that contains skimmed milk power by the freeze-drying liquid in the plate.
6. determining enrofloxacin content kit in the detection by quantitative food according to claim 1, it is characterized in that: described connector ENF-HRP is with Enrofloxacin, N-hydroxy-succinamide, N, N '-dicyclohexylcarbodiimide is dissolved in N, in the dinethylformamide, 360 ° of room temperatures of aluminium film parcel were shaken the centrifuging and taking supernatant 4～6 hours; Horseradish peroxidase HRP is dissolved in the sodium bicarbonate solution, under ice bath, be added dropwise to Enrofloxacin haptens reactant liquor in the HRP solution, under dripping fully back 4 ℃ 360 ° of reactant liquors are shaken up reaction and spend the night, at last all reactant liquors are put into the phosphate buffer dialysis 72h of bag filter with 0.01mol/L.
7. the detection method of the kit of determining enrofloxacin content in the detection by quantitative food as claimed in claim 1, it is characterized in that: detection method may further comprise the steps:
(1). when temperature is 10～30 ℃, carry out sample pre-treatments, obtain sample extracting solution;
(2). open kit and take out bag by plate, Enrofloxacin titer and sample extracting solution with various concentration joins bag by in the plate micropore separately respectively, and the parallel application of sample of diplopore, and then in various concentration Enrofloxacin titers and sample extracting solution micropore separately, add enzyme-labelled antigen solution, concussion mixing 2～8min, room temperature reaction 0.8～1.5 hour;
(3)., by plate 3～4 times liquid in the hole is got rid of with cleansing solution wash bags, buckle with thieving paper and do, add the mixed liquor of substrate A and substrate B, reacted under the room temperature 0.4～0.6 hour;
(4). in each micropore, add stop buffer, cessation reaction, read absorbance with microplate reader, draw the canonical plotting of Enrofloxacin according to the absorbance of various concentration Enrofloxacin titers, the canonical plotting of contrast Enrofloxacin obtains contents of enrofloxacin in the sample extracting solution.
8. the detection method of determining enrofloxacin content kit in the detection by quantitative food according to claim 7 is characterized in that: the proportioning of the mixed liquor of described substrate A and substrate B is 14.6: 0.45.
9. the detection method of determining enrofloxacin content kit in the detection by quantitative food according to claim 7 is characterized in that: sample thief adds extract, and oscillator mixing 2min is centrifugal, gets the part supernatant and dilutes with phosphate buffer.
10. the detection method of determining enrofloxacin content kit in the detection by quantitative food according to claim 9 is characterized in that: described extract is an acetonitrile: 0.1M NaOH is 4: 1 mixed solution by volume.
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| CNA2007101502838A CN101162230A (en) | 2007-11-22 | 2007-11-22 | Kit for quantitative determining enrofloxacin content in food product and testing method thereof |
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| CN102135534A (en) * | 2010-08-31 | 2011-07-27 | 华南农业大学 | Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof |
| CN102353785A (en) * | 2011-07-06 | 2012-02-15 | 清华大学深圳研究生院 | Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof |
| CN102507945A (en) * | 2011-12-05 | 2012-06-20 | 河北省科学院生物研究所 | Sulfamethazine enzyme-linked immunoassay kit |
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102135534A (en) * | 2010-08-31 | 2011-07-27 | 华南农业大学 | Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof |
| CN102353785A (en) * | 2011-07-06 | 2012-02-15 | 清华大学深圳研究生院 | Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof |
| CN102353785B (en) * | 2011-07-06 | 2013-11-06 | 清华大学深圳研究生院 | Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof |
| CN102507945A (en) * | 2011-12-05 | 2012-06-20 | 河北省科学院生物研究所 | Sulfamethazine enzyme-linked immunoassay kit |
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
| CN113721024A (en) * | 2021-09-16 | 2021-11-30 | 天津温阳生物技术有限公司 | Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food |
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