CN1811453A - Animal source food animal medicine residue detection reagent kit and detecting method thereof - Google Patents
Animal source food animal medicine residue detection reagent kit and detecting method thereof Download PDFInfo
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- CN1811453A CN1811453A CN 200610023659 CN200610023659A CN1811453A CN 1811453 A CN1811453 A CN 1811453A CN 200610023659 CN200610023659 CN 200610023659 CN 200610023659 A CN200610023659 A CN 200610023659A CN 1811453 A CN1811453 A CN 1811453A
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Abstract
The present invention provides a kit for detecting residue streptomycin content in zoonotic food and veterinary medicine and its detection method, belonging to the field of agricultural product detection and analysis technology. Said kit includes kit body, antigen or antibody correspondent to the residue in zoonotic food and veterinary medicine which can be detected by said kit, precoating plate or enzyme-labelled plate in which the correspondent monoclonal antibody is coated and avidin enzyme label. Besides, said invention also provides the concrete steps of said detection method.
Description
Technical field
The present invention relates to animal source food animal medicine residue detection reagent kit and detection method thereof in the agricultural product check and analysis technical field.
Background technology
China is agricultural product production and consumption big country, and wherein animal derived food occupies a big chunk ratio of agricultural product, and the detection of animal source food animal medicine residue is directly connected to resident's food consumption safety.
For example the streptomysin in the animal medicine residue (streptomycin) is an aminoglycosides antibiotics, be to be formed by connecting by oxo bridge by the aglycon that two or three amino sugar molecules and aglucone divide, streptomysin has the effect of powerful anti-Gram-negative bacteria, so be widely used in the treatment of Animal diseases.But streptomysin has the toxicity of a lot of aspects, one: can damage the glycometabolism and the energy utilization of inner ear organ of Corti internal and external hair cells, cause potassium on the inner ear hair cells film, sodion pump generation obstacle, and make the undermined ototoxicity that causes of hair cell function; Two: owing to streptomysin is accumulated through renal excretion with in cortex renis and caused renal toxicity; Three: streptomysin can also combine with the presynaptic membrane calcium binding site, stops calcium ion to participate in the neurotoxicity that release caused of acetylcholine.It has ototoxicity, neurotoxicity and Toxicity of Kidney.And for example the chloromycetin in the veterinary drug is a kind of broad-spectrum antibiotic, has good antibiotic and pharmacological property, is widely used in the control of the various infectious diseases of all kinds of poultry, domestic animal, aquatic animal (fish, shrimp etc.) and honeybee etc.But chloromycetin has serious adverse to the people, can cause alpastic anemia; Suchlike veterinary drug residual in food can influence human health, the use that all requires to limit the quantity of of country such as American-European-Japanese and China; But because this type of veterinary drug price comparison is cheap, result of treatment is better, the economy return height, and illegal use is still very general.Therefore strengthen the detection of animal derived food is very important.
At present, mainly adopt the residual quantity of instrumental method detection streptomysin, as thin layer electrophoresis (TLC), high pressure lipuid chromatography (HPLC) (HPLC), gas-matter online (GC/MS), liquid-matter online (HPLC/MS), Capillary Electrophoresis (CE) etc.Because complicated instrument and equipment and loaded down with trivial details process, be not suitable for the screening of on-site supervision and great amount of samples, wherein the ELISA detection method is a kind of novel being used as streptomysin residue detection screening technique in the animal derived food, as Chinese patent application (03206171.4) streptomysin ELISA detection kit, this patented claim is made of the titer and the cover plate film of 96 hole polystyrene elisa plates, 40ml concentrated cleaning solution, 11ml peroxidase labelling thing, 6ml streptomysin antibody, 6ml colour developing liquid A liquid, 6ml colour developing liquid B liquid, 6ml stop buffer, 6 bottles of variable concentrations.Adopt the indirect competitive ELISA method, on capillary strip, wrap in advance by coupled antigen, residue streptomysin in the sample will be competed anti-streptomycin antibody with the coupled antigen of pre-bag quilt on the capillary strip, after adding ELIAS secondary antibody, develop the color with tmb substrate, the sample light absorption value becomes negative correlation with the content of its contained residue streptomysin, relatively can draw the content of corresponding residue streptomysin with typical curve.Though this method compare with instrument analysis technology have quick, easy, characteristics, what but it adopted is two antienzyme technology and indirect competition immunologic detection method, wherein in two antienzyme technology, two anti-enzymes and a resistive connection close reaction, the site cross-cutting issue, easily cause non-specific binding, a little less than causing causing the relatively poor and indirect competition immunologic detection method less stable of sensitivity, antijamming capability in conjunction with deviation.
Summary of the invention
The purpose of this invention is to provide a kind of animal source food animal medicine residue detection reagent kit and detection method thereof; Adopt the direct competitive immune response of antigen and antibody in the present invention and adopt biotin-Avidin to amplify reactive system; Specificity is stronger, and sensitivity is higher, and stability is better, and antijamming capability is stronger.
Above-mentioned technical matters of the present invention is achieved by the following technical programs: a kind of animal source food animal medicine residue detection reagent kit, comprise box body, it is characterized in that this kit comprises that also pairing antigen of animal source food animal medicine residue or antibody that biotinylation detects with this kit and the pre-bag that is coated with corresponding monoclonal antibody are by plate or ELISA Plate and Avidin enzyme labeling thing.
Wherein said monoclonal antibody is generally mouse source antibody, and biotin is a kind of biotin, and Avidin is a kind of alkaline glycoprotein that is present in the egg white, Avidin molecule can with 4 biotin molecule stable bond.Avidin can combine with protein (comprising antigen, antibody, enzyme etc.) molecule with biotin and not influence the biologically active of Avidin and biotin, is desirable marking agent.But antigen molecule coupling dozens of biotin and Avidin molecule, and Avidin or biotin molecule can combine with enzyme, thus form a molecule amplification system, significantly improve the sensitivity of detection.
In above-mentioned animal source food animal medicine residue detection reagent kit, the horseradish hydrogen peroxidase of described Avidin marker enzyme Avidin mark.
For more convenient on-site supervision and great amount of samples examination, in animal source food animal medicine residue detection reagent kit, described kit also comprises standard solution, biotinylated antigen dilution, developer, stop buffer, the concentrated cleaning solution of animal source food animal medicine residue.
In above-mentioned animal source food animal medicine residue detection reagent kit, described biotinylated antigen dilution is the phosphate buffer that contains polyglycol 20,000.
In above-mentioned animal source food animal medicine residue detection reagent kit, described developer is made up of developer A and developer B, described developer A is the citrate buffer that contains absolute ethyl alcohol, urea peroxide, and described developer B is for containing the tetramethyl benzidine citrate buffer.
In above-mentioned animal source food animal medicine residue detection reagent kit, described stop buffer is 2M H
2SO
4
In above-mentioned animal source food animal medicine residue detection reagent kit, described concentrated cleaning solution is the phosphate buffer of 1% tween.
A kind of detection method of animal source food animal medicine residue detection reagent kit: may further comprise the steps:
1, the pairing monoclonal antibody of a kind of animal source food animal medicine residue that pre-bag quilt detects with this kit on wrapping by plate in advance or on the title capillary strip;
2, the standard items of corresponding animal source food animal medicine residue or the residue in the sample and corresponding biotinylated antigen, the two and antibody competition react, and form antibody-biotinylated antigen compound;
3, the Avidin that adds enzyme labeling, the compound of formation antibody-bioid antigen-Avidin;
4, tmb substrate colour developing.The light absorption value of sample becomes negative correlation with the content of its contained residue streptomysin, compares with typical curve, can draw the content of corresponding residue streptomysin.
The present invention has the following advantages:
1, the scope of application is wider: be applicable to the detection of various animal source food animal medicine residue residual quantities such as aquatic products, honey, feed, milk and goods, animals urine, muscle, tissue.
2, the present invention's characteristics such as having quick, easy, accurate, strong interference immunity and sensitivity height of comparing with instrument analytical method, the running time only must 1.5 hours, detect lower limit and reach below the 0.05ppb, can reduce operate miss and working strength to greatest extent.
3, the present invention adopts unique biological element-Avidin amplification system, has characteristics sensitiveer more, stable more than two traditional antienzyme technical methods, strong interference immunity.If the rapid screening when being used for the raw material examination of honey processing factory detects, honey directly can be diluted the back and detect, save a large amount of sample pre-treatment time and expense.
Description of drawings
Fig. 1 is the structural representation of animal source food animal medicine residue detection reagent kit of the present invention.
Fig. 2 is that 96 pore chain mycin monoclonal antibodies are wrapped in advance by the structural representation of plate in this animal source food animal medicine residue detection reagent kit.
Embodiment
The invention will be further described below to engage embodiment, but the present invention is not limited to these embodiment:
One, streptomysin residue detection kit and detection method thereof
Embodiment 1: biotinylation streptomysin antigen and streptomysin MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) biotinylation streptomysin antigen is synthetic
Antigen (antibody) dialysis under alkalescence (carbon delays PH9.6) condition adds activation biotin synthetic reaction with it more than eight hours again, in neutral (general phosphate buffer PH7.5) dialysis, removes unnecessary activation biotin at last.
(2) streptomysin Monoclonal Antibody
Immune mouse was got mouse spleen cell and myeloma cell and is merged more than two weeks, carried out the cell line colony screening, picked out the cell line of best secretory antibody.Above monoclonal antibody clonal antibody cell line, injection mouse peritoneal can obtain monoclonal antibody ascites.This cell line not the time spent frozen, the time spent recovers, cultivates.
Used monoclonal antibody all needs to carry out purifying through oversalting, molecular sieve and affinity chromatography and extracts.
Embodiment 2:
(1) structure of streptomysin residue detection kit
The structure of kit as depicted in figs. 1 and 2, mainly contain box body 1,96 pore chain mycin monoclonal antibodies are wrapped in advance by plate 2,6 bottles of streptomysin series concentration standard items 3, biotinylation streptomysin antigen working fluid 4, biotinylated antigen dilution 5, Avidin enzyme labeling thing working fluid 6, substrate developer A liquid 7, substrate developer B liquid 8, stop buffer 9, concentrated cleaning solution 10 and foam carriage, the foam carriage is provided with shrinkage pool, above-mentioned 3-10 is placed in the shrinkage pool of foam carriage, foam carriage and bag are placed in the box body 1 by plate 2, and wherein the pre-bag of 96 pore chain mycin monoclonal antibodies is made up of capillary strip by plate 2.
(2) preparation of agents useful for same
A. streptomysin standard solution: 6 bottles of streptomysin series standard solution, 1～3ml/ bottle
B. biotinylation streptomysin antigen working fluid, 50% glycerite;
C. biotinylated antigen dilution contains the phosphate buffer of polyglycol 20,000;
D. Avidin enzyme labeling thing working fluid contains the albuminous phosphate buffer of ox blood;
E. substrate developer A liquid contains the citrate buffer of absolute ethyl alcohol urea peroxide;
F. substrate developer B liquid contains the tetramethyl benzidine citrate buffer;
G. stop buffer, 2M H
2SO
4
H. concentrated cleaning solution, the phosphate buffer of 1% tween
I. normal hexane;
J.PBST damping fluid preparation (extract and use): 1.15g Na
2HPO
40.2g kH
2PO
40.2gKCl 9g NaCl; 0.5ml Tween-20 adding distil water or deionized water 1000ml are the PBST damping fluid.
K. extract damping fluid: 1g heptane-sulfonate sodium (M=202.2), 1.5gNa
2HPO
412H
2O.Add distilled water to 100ml, add the 0.75ml strong phosphoric acid and transfer PH to 2.0.
(3) 96 pore chain mycin monoclonal antibodies are wrapped in advance by the preparation of plate
The phosphate buffer (PH9.6) that contains monoclonal antibody, bag are added confining liquid (containing the albuminous phosphate buffer of ox blood) sealing, freeze-drying, vacuum packaging by more than 24 hours.
(4) used instrument
---microplate reader,
---homogenizer,
---oscillator,
---hydro-extractor,
---micro sample adding appliance: single track 20ul-200ul, 200ul-1000ul, multiple tracks 250ul.
The pre-treatment of embodiment 3, sample
(1) milk sample: get milk 2ml, it is put the centrifugal 10min of 5000rpm, remove upper strata fat; Take off a layer 50ul, press 1: 40 times of dilution with the PBS damping fluid.(the 1950ulPBS damping fluid adds 50ul milk) diluted sample multiple: 40 times.
(2) chicken meat sample: 2g removes the pulverizing sample and 8ml PBST damping fluid mixing 5min of fat, and adding normal hexane 5ml mixes 10min fully up and down and leaves standstill 1h.The centrifugal 15mim of room temperature, 10000rpm; Get middle layer 1ml with pipettor, add the 1ml normal hexane, 5min fully vibrates; The centrifugal 15min of room temperature, 4000rpm; Remove the upper strata, take off layer with 1: 10 times of dilution (450ul PBS damping fluid adds the 50ul supernatant); Diluted sample multiple: 40 times.
(3) liver sample: 5.0g removes the pulverizing sample and 20ml PBST damping fluid mixing 30min of fat; The centrifugal 10min of room temperature, 10000rpm; The 2ml supernatant mixes vibration 5min with the 3ml normal hexane; The centrifugal 10min of room temperature, 4000rpm; Remove the upper strata fat deposit, diluted (the 900ul dilution adds the 100ul supernatant) with 1: 10 with the PBS damping fluid after the dilution; Getting the 60ul water analyzes; Diluted sample multiple: 40 times.
(4) blood serum sample: get the blood serum sample 20ul after centrifugal, with the PBS damping fluid by dilution (3980ul PBS damping fluid adds 20ul serum) in 1: 200; Diluted sample multiple: 200 times;
(5) honey product sample
Residual streptomysin in embodiment 4, the test sample
(1) takes by weighing weighing 1g sample and add the extraction damping fluid to 10ml; Vibration 10min dissolves fully, and the centrifugal 10min/4000rpm of room temperature is until limpid; With RIDA C18 post (purified extract
(2) wash pillar with 2ml methyl alcohol (100%), flow velocity is 60d/min;---wash pillar with 2ml, flow velocity is 60d/min; Last sample is got the 2ml sample, and flow velocity is 15d/min; With nitrogen or air blow drying pillar, 3min.(removing moisture content in the post); With 1ml methyl alcohol (100%) elution samples, flow velocity is 15d/min; At 40-50 ℃, weak air or nitrogen flow down complete evaporating solvent; With the dry residue of PBS damping fluid dissolving after the 2ml dilution, get 60ul and analyze; Extension rate: 10 times; Detect lower limit: 3ppb.Wherein above all reagent and sample preparation must be carried out under the room temperature
(3) sample detection
All reagent and sample must be put room temperature (about 20 ℃ ± 5 ℃) before test experience, kit is opened, and balance is more than 1 hour; Take out framework and need the capillary strip of quantity, no capillary strip is put into valve bag, be stored in (2-8 ℃); According to institute's expense biotinylated antigen is diluted (1 part add 19 part dilutions) with dilution at 1: 20, only use for existing the detection.(biotinylated antigen of not diluted is in-20 ℃ of preservations); Application of sample: establish blank well one hole, add PBS or physiological saline 100ul; Standard solution and testing sample are carried out the parallel application of sample 50ul of diplopore, add biotinylation streptomysin antigen 50ul simultaneously; Shrouding vibration mixing 1 minute was inserted 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 30 minutes; Wash plate machine washing plate 5 times.Wash plate 3 times by hand: liquid in the hole is got rid of, buckle with thieving paper and do, add washing lotion 250ul/ hole, left standstill 1 minute, get rid of liquid again, button is done.Repetitive operation 3 times; Every hole adds enzyme labeling Avidin solution 100ul (blank well adds PBS or physiological saline 100ul), and shrouding was inserted 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 30 minutes; Wash plate machine washing plate 5 times, wash plate 3 times by hand, each 1 minute at interval; Add developer A, each 50ml of developer B, shrouding mixing 15 seconds, 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 15 minutes; Every hole adds stop buffer 50ul, and the mixing that vibrates gently reads 450nm light absorption value (make zero light absorption value with blank well, suggestion detects with dual wavelength 450/630nm, add stop buffer run through data in 15 minutes).
Reagent used in the present embodiment is prepared according to the reagent compound method among the embodiment 2.
The mean value of standard that is obtained and sample absorbance multiply by 100 again divided by the absorbance of first standard (0 standard).Therefore 0 standard equals 100% and provide light absorption value degree value with number percent.
It is the semilog coordinate system curve map of a corresponding streptomysin concentration (ug/kg) that the standard value of calculating plots, and calibration curve is worked as 0.3～24.3ug/kg (ppb) scope planted agent becomes linearity.The concentration of corresponding each sample (ug/kg) can be read from calibration curve.
The item that streptomysin kit of the present invention is in use noted:
1, room temperature is lower than 20 ℃ or reagent and sample and does not get back to room temperature (about 25 ℃) and can cause the OD value of all standards on the low side.
2, in washing the plate process if the plate hole dry situation, can be accompanied by then that typical curve to occur non-linear, the phenomenon that repeatability is bad is so carry out next step operation immediately after washing plate and patting dry.
3, mix and will evenly wash plate and want thoroughly, otherwise can the bad phenomenon of duplicating property.
4, reaction terminating liquid is a 2M sulfuric acid, avoids contacting skin.
5, condition of storage: kit is put no capillary strip into valve bag and is resealed in 2 ℃ of-8 ℃ of preservations; Because standard substance and colourless colour former must be avoided being directly exposed under the high light to photaesthesia.
6, the rotten phenomenon of reagent
Colour developing liquid is rotten if there is any color to show, should abandon it.The absorbance of 0 standard is less than 0.8 (A of unit
450＜0.8) time, expression reagent may go bad.
7, add that general colour developing 15min get final product behind A, the B liquid, more shallow as if color, but the proper extension developing time, but can not surpass 20min.
8, must not use reagent constituents after the failure period, the different batches reagent constituents must not be mixed use.
9, each plate hole all is used to measure optical curve at last, suffers inside and outside should preventing at the bottom of the hole stained.
(1) residual chloromycetin quality testing test agent box is formed:
A. chloromycetin standard solution: 6 bottles of chloromycetin series standard solution, 1～3ml/ bottle
B. biotinylation chloramphenicol antibody working fluid,
C. biotinylated antibody dilution,
D. Avidin enzyme labeling thing working fluid,
E. substrate developer A liquid,
F. substrate developer B liquid,
G. stop buffer,
H. concentrated cleaning solution,
(2) 96 hole pre-coated elisa plates
The preparation method of above material is with embodiment 2
(3) used instrument is with embodiment 2, reagent
Reagent:
Below all reagent all recommend operational analysis pure or top grade is pure; Water is distilled water or goes
Ionized water.
---ethyl acetate
---acetonitrile
---normal hexane
---sodium nitroprusside
---zinc sulfate
The pre-treatment of embodiment 6, sample
Concentration and dilution liquid was diluted (being used for the dilution of antibody and the dilution after the sample extraction) with distilled water by 1: 10
(a) tissue (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method: with homogenizer homogeneous structure sample; Sample behind title 3g ± 0.1g homogeneous adds 6ml ethyl acetate in centrifuge tube, vibration 10min, and more than the room temperature 3000g, centrifugal 10min; Take out 4ml supernatant liquid (sample that is equivalent to 2g approximately) and flow down 50～60 ℃ of dryings at nitrogen; The residue that adds 1ml n-hexane dissolution drying adds the 1ml dilution 1min that vibrates strongly again, more than the room temperature 3000g, and centrifugal 5min; Remove upper strata normal hexane phase, take off layer water 50 μ l and analyze; Diluted sample multiple: 0.5 times.
(b) serum blood plasma pre-treating method: get 1ml serum or blood plasma to test tube, add 2ml ethyl acetate vibration 1min; Leave standstill and make water and organic phase layering or room temperature 3000g, centrifugal 5min; The ethyl acetate that pipettes the upper strata is to another test tube, with drying up in 50 ℃ of water-baths of nitrogen stream; Residue dissolves with the 1ml dilution; Getting 50 μ l analyzes; Annotate: the sample extension rate is 1 times.
(c) urine pre-treating method: pipette the 2ml urine in centrifuge tube, add pH4.8100mM sodium-acetate buffer 0.5ml and mix; (Merck is Art.No.4114) in the urine of dilution, in 37 ℃ of hydrolysis at least 2 hours (or spending the night) to add the 40ul glucuronidase; This solution adds 8ml ethyl acetate mixing 1min after returning to room temperature; The centrifugal 10min of room temperature 3000g takes out 4ml supernatant liquid (being equivalent to the 0.5ml urine sample) 50～60 ℃ of dryings under nitrogen; Getting 50 μ l with the dry residue of 1ml dilution dissolving analyzes; Annotate: the sample extension rate is 1 times.
(d) honey pre-treating method: get 2g honey, put into centrifuge tube, use the 4ml deionized water dissolving; Add 4ml ethyl acetate and shake 10min up and down; The above centrifugal 10min of room temperature 3000g; Pipette 1ml upper strata ethyl acetate (being equivalent to the 0.5g sample) in another centrifuge tube, 50～60 ℃ of nitrogen flow down evaporate to dryness; Residue dissolves with the 0.5ml dilution after diluting; Getting 50ul analyzes; The diluted sample multiple is 1 times.
(e) casing pre-treating method: the sample behind title 1g ± 0.1g homogeneous adds 10ml ethyl acetate in centrifuge tube, vibration 10min, and more than the room temperature 3000g, centrifugal 10min; Take out 5ml supernatant liquid (sample that is equivalent to 0.5g) and flow down 50～60 ℃ of dryings at nitrogen; The residue that adds 1ml n-hexane dissolution drying adds the 0.5ml dilution 1min that vibrates strongly again, more than the room temperature 3000g, and centrifugal 5min; Remove the upper strata phase, take off layer water 50 μ l and analyze; Diluted sample multiple: 1 times.Annotate: dry sample originally need shred after (the not super 5mm of length) homogeneous again, and wet sample need above with rinsed with deionized water 20min (removing surperficial salt), carry out homogeneous after draining again.
(f) milk and milk powder pre-treating method:
Need the liquid of preparation
C liquid: 0.36M sodium nitroprusside (Na2Fe (CN) 5NO2H2O): take by weighing the 10.7g sodium nitroprusside, be dissolved in the 100ml deionized water.
D liquid: 1M zinc sulfate (ZnSO47H2O): take by weighing 28.8g zinc sulfate, be dissolved in the 100ml deionized water.
Milk sample disposal route one: milk sample, 10 ℃ of above centrifugal 10min of 3000g absorb upper strata fat; Get 5ml and remove the fat milk sample to centrifuge tube, add 150ul C liquid and precipitation occurs, add 150ul D liquid after the short term oscillation and mix; 15 ℃ of above centrifugal 10min of 3000g pipette upper strata liquid; Dilute upper strata liquid with dilution with equal-volume; Getting 50ul analyzes; Attention:, repeat precipitation process again if still muddy after centrifugal.Extension rate is 2.1 times.
Milk sample disposal route two: get 5ml and remove the fat milk sample to centrifuge tube; Add 250ul C liquid and 250ul D liquid and thoroughly mix, the 4-12 ℃ of above centrifugal 10min of 3000g.If there is not refrigerated centrifuge, please sample is cooled to 8 ℃ in advance; Migrate out 2.2ml upper strata liquid (be equivalent to 2ml milk sample) to a new centrifuge tube, add the 4ml ethyl acetate 10min that vibrates up and down; The above centrifugal 10min of room temperature (20-25 ℃) 3000g; Migrate out 2ml ethyl acetate supernatant liquid (being equivalent to 1ml milk sample), 60 ℃ of nitrogen flow down bone dry; With the dry residue of 0.5ml dilution dissolving; Getting 50ul analyzes; Extension rate is 0.5 times.
Powdered milk sample disposal route: claim 2g milk powder to centrifuge tube, add the 10ml deionized water, the vibration dissolving; Add 1ml C liquid and 1ml D liquid thoroughly mixes.4-12 ℃ of 3000g is above from 10min.If no refrigerated centrifuge please is cooled to sample 8 ℃ in advance; Migrate out 3.6ml upper strata liquid (being equivalent to 0.6g milk powder) to a new centrifuge tube, add the 6ml ethyl acetate 10min that vibrates back and forth up and down; The above centrifugal 10min of room temperature (20-25 ℃) 3000g; Migrate out 4ml ethyl acetate supernatant liquid (being equivalent to 0.4g milk powder), 60 ℃ of nitrogen flow down bone dry; With the dry residue of 0.4ml dilution dissolving; Getting 50ul analyzes; Extension rate is 1 times.
Eggs pre-treating method: with the equal quality sample of homogenizer low speed (yolk or shell egg); Take by weighing the sample that the 3g homogeneous is crossed, with 9ml acetonitrile-aqueous solution (84+16; V+V) 10min is extracted in vibration, and 15 ℃ of 3000g are above from 10min; Get 3ml upper strata liquid and mix, add 4.5ml ethyl acetate mixing 5min, 15 ℃ of above centrifugal 10min of 3000g with 3ml distilled water; Upper organic phase transferred in the test tube dry up with nitrogen; After adding 1ml n-hexane dissolution residue, mix 1min, centrifugal removal normal hexane phase with the reextraction of 2ml dilution; Getting 50ul lower floor analyzes mutually; Annotate: the sample extension rate is 2.
Sample detection by quantitative lower limit
Chicken/chicken gizzard, pork/pork liver, shrimp, fish----------0.05ppb
Honey----------------------------------0.15ppb
Urine, serum----------------------------0.1ppb
Casing----------------------------------0.1ppb
Milk----------------------------------0.05ppb
Eggs----------------------------------0.1ppb
Residual chloromycetin in embodiment 7, the test sample
1, all reagent and sample must be put room temperature (about 20 ℃ ± 5 ℃) before test experience, and kit is opened, and balance is more than 1 hour.
2, take out framework and need the capillary strip of quantity, no capillary strip is put into valve bag, be stored in (2-8 ℃).
3, according to institute's expense biotinylated antibody is diluted (1 part add 19 part dilutions) with the biotinylated antibody dilution at 1: 20, only use for existing the detection.(biotinylated antibody of not diluted is in-20 ℃ of preservations).
4, application of sample: establish blank well one hole, add PBS or physiological saline 100ul; Standard solution and testing sample are carried out the parallel application of sample 50ul of diplopore, add biotinylation chloramphenicol antibody 50ul simultaneously; Shrouding vibration mixing 1 minute was inserted 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 30 minutes.
5, wash plate machine washing plate 5 times.Wash plate by hand: liquid in the hole is got rid of, buckle with thieving paper and do, add washing lotion 250ul/ hole and left standstill 1 minute, get rid of liquid again, button is done.Repeat this time to operate 3 times.
6, every hole adds enzyme labeling Avidin solution 100ul (blank well adds PBS or physiological saline 100ul), and shrouding was inserted 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 30 minutes.
7, wash plate machine washing plate 5 times, wash plate 3 times by hand, each 1 minute at interval.
8, add developer A, each 50ml of developer B, shrouding mixing 15 seconds, 37 ℃ of constant incubators or 37 ℃ of constant water bath box incubations 15 minutes.
9, every hole adds stop buffer 50ul, and the mixing that vibrates gently reads 450nm light absorption value (make zero light absorption value with blank well, suggestion detects with dual wavelength 450/630nm, add stop buffer run through data in 15 minutes).
The mean value of standard that is obtained and sample absorbance (OD) value multiply by 100 again divided by the OD value of first standard (0 standard).Therefore 0 standard equals 100% and provide the OD value with number percent.
The standard value of calculating plots the semilog coordinate system curve map of a corresponding chloramphenicol concentration (ug/kg), and calibration curve is worked as 0.05～4.05ug/kg (ppb) scope planted agent becomes linearity.The concentration of corresponding each sample (ug/kg) can be read from calibration curve.
Specific embodiment described in the present invention only is that the present invention's spirit is illustrated.The technician of the technical field of the invention can make various modifications or replenishes or adopt similar mode to substitute described specific embodiment, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Although the present invention has been made detailed explanation and has quoted some instantiations as proof, to those skilled in the art, only otherwise leave that the spirit and scope of the present invention can be done various variations or correction is obvious.
Claims (9)
1, a kind of animal source food animal medicine residue detection reagent kit, comprise box body, it is characterized in that this kit comprises that also pairing antigen of animal source food animal medicine residue or antibody that biotinylation detects with this kit and the pre-bag that is coated with corresponding monoclonal antibody are by plate or ELISA Plate and Avidin enzyme labeling thing.
2, animal source food animal medicine residue detection reagent kit according to claim 1, it is characterized in that, described monoclonal antibody is generally mouse source antibody, biotin is a kind of biotin, Avidin is a kind of alkaline glycoprotein that is present in the egg white, Avidin molecule can with 4 biotin molecule stable bond.
3, animal source food animal medicine residue detection reagent kit according to claim 1 is characterized in that, the horseradish hydrogen peroxidase of described Avidin marker enzyme Avidin mark.
4, according to claim 1 or 2 or 3 described animal source food animal medicine residue detection reagent kits, it is characterized in that described kit also comprises standard solution, biotinylated antigen dilution, developer, stop buffer, the concentrated cleaning solution of animal source food animal medicine residue.
5, animal source food animal medicine residue detection reagent kit according to claim 1 is characterized in that, described biotinylated antigen dilution is the phosphate buffer that contains polyglycol 20,000.
6, animal source food animal medicine residue detection reagent kit according to claim 4, it is characterized in that, described developer is made up of developer A and developer B, described developer A is the citrate buffer that contains absolute ethyl alcohol, urea peroxide, and described developer B is for containing the tetramethyl benzidine citrate buffer.
7, animal source food animal medicine residue detection reagent kit according to claim 4 is characterized in that, described stop buffer is 2M H
2SO
4
8, animal source food animal medicine residue detection reagent kit according to claim 4 is characterized in that, described concentrated cleaning solution is the phosphate buffer of 1% tween.
9, a kind of detection method of animal source food animal medicine residue detection reagent kit: may further comprise the steps:
(1), the pairing monoclonal antibody of a kind of animal source food animal medicine residue that pre-bag quilt detects with this kit on wrapping by plate in advance or on the title capillary strip;
(2), standard items or the residue in the sample and the corresponding biotinylated antigen of corresponding animal source food animal medicine residue, the two and antibody competition reaction form antibody-biotinylated antigen compound;
(3), add the Avidin of enzyme labeling, form the compound of antibody-bioid antigen-Avidin;
(4), tmb substrate colour developing: the light absorption value of sample becomes negative correlation with the content of its contained residue streptomysin, compares with typical curve, can draw the content of corresponding residue streptomysin.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610023659 CN1811453A (en) | 2006-01-26 | 2006-01-26 | Animal source food animal medicine residue detection reagent kit and detecting method thereof |
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| CN 200610023659 CN1811453A (en) | 2006-01-26 | 2006-01-26 | Animal source food animal medicine residue detection reagent kit and detecting method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520190A (en) * | 2011-12-15 | 2012-06-27 | 中国人民解放军总医院 | Enzyme-linked immunoassay detection reagent kit for human insulin-like growth factor II |
| CN107389958A (en) * | 2017-08-30 | 2017-11-24 | 深圳大学 | The detection gel column of lincomycin and the detection method of lincomycin |
| CN113156126A (en) * | 2021-03-05 | 2021-07-23 | 清远海贝生物技术有限公司 | ELISA kit for detecting vomitoxin and detection method thereof |
-
2006
- 2006-01-26 CN CN 200610023659 patent/CN1811453A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520190A (en) * | 2011-12-15 | 2012-06-27 | 中国人民解放军总医院 | Enzyme-linked immunoassay detection reagent kit for human insulin-like growth factor II |
| CN102520190B (en) * | 2011-12-15 | 2014-03-05 | 中国人民解放军总医院 | Enzyme-linked immunoassay detection reagent kit for human insulin-like growth factor II |
| CN107389958A (en) * | 2017-08-30 | 2017-11-24 | 深圳大学 | The detection gel column of lincomycin and the detection method of lincomycin |
| CN113156126A (en) * | 2021-03-05 | 2021-07-23 | 清远海贝生物技术有限公司 | ELISA kit for detecting vomitoxin and detection method thereof |
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