CN100516880C - Detecting method for quick detecting veterinary medicinal residue in food and agriculture and sideline products - Google Patents
Detecting method for quick detecting veterinary medicinal residue in food and agriculture and sideline products Download PDFInfo
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- CN100516880C CN100516880C CNB2005100264871A CN200510026487A CN100516880C CN 100516880 C CN100516880 C CN 100516880C CN B2005100264871 A CNB2005100264871 A CN B2005100264871A CN 200510026487 A CN200510026487 A CN 200510026487A CN 100516880 C CN100516880 C CN 100516880C
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Abstract
The present invention belongs to the field of toxic matter detecting technology. The detection method of quickly detecting veterinary medicinal residue in food and agriculture and sideling products includes the following steps: applying coupled antigen of several kinds of veterinary medicines as micro array on solid carrier and coating; mixing deluent diluted biotinylated antibody and measured sample, competitive immunogical reaction with the coated veterinary medicinal antigen, and washing with detergent; adding avidin marked colloidal gold for amplification and colorizing, and obtaining grey result with scanning instrument. The competitive immunogical method of the present invention has the features of high sensitivity, high specificity and simple operation.
Description
Technical field
The invention belongs to the technical field that noxious material detects, relate in particular to a kind of detection method that detects agricultural byproducts, food veterinary drug residue thing.
Background technology
China is agricultural product production and consumption big country, and the detection of agricultural product veterinary drug residue thing is directly connected to agricultural products in China production, outlet and resident's food consumption safety.Particularly the agricultural products in China outlet meets with the foreign technology trade barrier in recent years continuously because of animal medicine residue, and only agricultural product outlet in a year in Zhejiang Province's is prohibited more than one hundred million dollars.In recent years, food-safety problem has become one of key content of country's concern, because of the exceed standard food pollution incident that causes and the outlet of poisonous residuals is obstructed and taken place again and again, caused the great attention of national departments concerned, and having started action plan and market for farm products access systems such as non-polluted farm product and food security continuously, Fast Detection Technique and the product of developing various objectionable impuritiess in the food become very important demand.
Detect the method for food veterinary drug residue, mainly contain micro-biological process, physico-chemical analysis method (chromatography, application of gas chromatorgraphy/mass, gas chromatography mass spectrometry method), immunological method (radioimmunology, euzymelinked immunosorbent assay (ELISA)) etc.The microorganism detection method expense is cheap, but complicated operation is time-consuming, and the result judges by accident easily, poor specificity.Immunological method (enzyme is exempted from method) has more application in recent years, easy, the highly sensitive characteristics of basic symbols closing operation, and had relevant kit commodity to use.The physico-chemical analysis method is mainly used to confirmatory test, needs expensive instrument supporting.This patented claim relates to a kind of method of many indexs parallel detection albumen as Chinese patent application (01105023.3), comprise: will combine a plurality of can the contact with the liquid to be measured that contains target protein A, and form stable compound B-A with the solid phase carrier of the protein B of index specificity combination in the body; Further with the polyprotein mixed liquor in the specific bond PROTEIN C-labelled reaction of A of correspondence markings, form and stablize B-A-C-labelled, mark is the label of chemoluminescence method; Produce chemiluminescence reaction and input.This patented claim also provides a kind of methods of making protein chips, and is used for protein chip, the kit of the parallel detection of many indexs.This patented claim mainly is a principle of continuing to use enzyme linked immunosorbent assay (ELISA) (ELISA), exists that specificity is weak, sensitivity is low, range of application is narrow, the shortcoming of complicated operation.
And for example Chinese patent application (02137940.8) relates to a kind of chloromycetin (CAP) enzyme immunoassay kit and detection method thereof, belongs to the EIA enzyme immunoassay technical field.This invention prepared kit, take enzyme immunoadsorption (ELISA) competition law to detect CAP, utilize the coupled thing of CAP and bovine serum albumin(BSA) (BSA) as CAP antigen immune mouse, acquisition contains the monoclonal CAP antiserum of CAP antibody, be coated on the ELISA Plate after separating, purify, diluting, incubation, washing add sample diluting liquid and enzyme-labelled antigen, make both react, wash, add the zymolyte colour developing.Though this patent is simple to sample pre-treatments, be a kind of easy, quick, sensitive, accurate, cheap detecting method, but because the limitation of itself must cause its check and analysis process can not realize serialization, integrated, microminiaturized, high-throughout requirement.
Summary of the invention
The present invention is directed to that there is poor specificity in the used detection method of prior art, sensitivity is low, range of application is narrow, the shortcoming of complicated operation, provide a kind of sample pre-treatments simple, detect easy, quick, sensitive, detect the method for residue of veterinary drug accurately.
Technical matters of the present invention solves by the following technical programs: the detection method of a kind of fast detecting food, agricultural byproducts veterinary drug residue thing may further comprise the steps:
(1), multiple veterinary drug coupled antigen is wrapped quilt according to microarray point on solid phase carrier and to it; Wherein fixing veterinary drug antigen on the solid-phase matrix;
(2), will mix through the biotinylated antibody of diluted and testing sample, with the immune response that is at war with of multiple veterinary drug envelope antigen fixing on the solid phase carrier, cleansing solution washing; If testing sample does not contain veterinary drug in the course of reaction, the then multiple veterinary drug envelope antigen combination of fixing on antibody and the solid phase carrier; If testing sample contains veterinary drug, then biotin labeled antibody can combine with the veterinary drug in the testing sample, and by wash-out;
(3), the collaurum that adds the Avidin mark amplifies and dye, by scanner acquisition gray scale result.
The present invention adopts immunity competition ratio juris: adopt immune competition law to detect multiple residue of veterinary drug, its principle of work be utilize in the sample residual animal medicine be fixed on veterinary drug coupled antigen on the carrier at the antibody immune response that is at war with, by the biotin-avidin amplification system and by nm of gold dyeing the residual animal medicine of denier is done quantitative test, wherein tested residual animal medicine all has identical binding ability with microbiotic on being fixed on matrix to the antibody that indicates biotin, so have in limited time at the antibody that indicates biotin, vying each other will appear in this combination, each other restriction.Concrete reaction equation is as follows:
P1 + Q*?＝ P1-Q*
(being fixed on the veterinary drug on the matrix) (indicating the antibody of biotin) (compound 1)
+
P2 (tested residual animal medicine)
‖
P2-Q* (compound 2)
In the antibody amount one that indicates biotin regularly, the veterinary drug and the tested residual animal medicine that are fixed on the matrix depend on the two concentration ratio with the amount that indicates the antibodies of biotin, the P1 binding capacity, to reduce along with the increase of P2, this has illustrated that tested residual animal medicine has suppressed to be fixed on combining of veterinary drug and the antibody that indicates biotin on the matrix.Owing to be marked with biotin on the antibody, can combine with Avidin, collaurum is arranged on the Avidin, obtain the gray scale result by scanner (CCD).The antibiotic content of the black more then testing sample of image is low more, otherwise, then high more!
In addition, can also obtain the effect of detection by quantitative,, and adopt the appropriate mathematic model match wherein by the original dose-response curve of investigating three indexs of standard sample.Relation between concentration and the gray-scale value meets the pattern of y=a (x) b+c or other functions, and wherein y and x represent index output concentration respectively and detect gray scale, and a and b, c are constants, and different indexs have a, b value separately.The related coefficient g2 of dose-response curve should be not less than 0.97, with basic parameters such as above-mentioned dose-response curve equation, kind, index quantity, indication informations, be arranged in the database file of software, with CD writer with this document imprinting on CD.During detection, data are called by software automatically by CD, promptly know concentration value by gray scale, the detection by quantitative antibiotic concentration, the serialization of check and analysis process, integrated, microminiaturized, high-throughout advantage have been realized, and detecting operation only needs 10 minutes, and collaurum result relatively fluorescence is stable, reduces environmental pollution.
In above-mentioned detection method, described is 2: 5～1: 6 through the biotinylated antibody of diluted and the blending ratio of sample, sending out the time of answering after wherein the biotinylated antibody of process diluted mixes with sample is 5～20min, and the temperature of reaction is 30 ℃～38 ℃.
Table 1: the optimization of biotinylated antibody and sample blending ratio
Show that by above result the reaction result gray difference of 0ppb and 6.25ppb significantly is that biotinylated antibody and sample blending ratio are 2: 5～1: 6.
Table 2: the mixed reaction time of biotinylated antibody and sample is to the influence of gray scale
Above result shows: grey value difference whole between 5～20min is not too big, so the incubation 5～20min that adopts, the heated culture temperature of employing is generally 37 ℃.But it is also little to handle whole grey value difference between 30 ℃～38 ℃.
The labeling method of the biotinylated antibody of wherein said process diluted is: with DMF BNHS is made into 0.8～1.2mg/mL solution, with 0.08～0.12mol/L, pH8.2～9.2NaHCO3 is 1～3mg/mL with the antibody purified dilution, volumetric ratio by BNHS: IgG is mixing in 1: 8～1: 15, react 1～5h under the stirring at room, the bag filter of packing into adds 0.01mol/L～1mol/L, pH7.0～7.6PBS solution, 4 ℃ of dialysed overnight.Add equal-volume glycerine in the bond, packing in a small amount ,-30 ℃ are frozen standby.
In above-mentioned detection method, the labeling method optimization of C of described biotinylated antibody is: with DMF BNHS is made into 1mg/mL solution, uses 0.1mol/L, pH9.0NaHCO
3Antibody purified dilution is 2mg/mL, is 1: 10 by the volumetric ratio of BNHS: IgG, reacts 2～4h under the stirring at room, the bag filter of packing into, adding 0.05mol/L, pH7.2PBS solution, 4 ℃ of dialysed overnight.
Wherein Avidin labeling method (sample cleansing solution prescription) is:
1. the pH with 0.08～0.12mol/L carbonate adjusting aurosol is 8.5～9.5;
2. adding (number percent by volume) in aurosol is 1%～4% protein solution, stirs 1～4 minute;
3. the concentration that adds (number percent by volume) 4.5%～5.5% is 0.8～1.2%PEG20000 solution;
4. be in the ultracentrifuge of 10000g～100000g centrifugal 30～60 minutes in rotating speed, inhale and remove supernatant;
5. precipitation is suspended in the damping fluid that contains 0.2～0.5mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 10000g～100000g, recover with same damping fluid again, concentration is advisable about with A 1cm/540nm=1.5, anticorrosion with 0.2～0.8mg/ml Sodium azide, put 4 ℃ of preservations.
In above-mentioned detection method, carbonate is sal tartari in the described Avidin labeling method step 1.
In above-mentioned detection method, described Avidin labeling method step 1 is: with 0.08mol/L～0.12mol/L K
2CO
3Regulate aurosol pH to 8.8～9.2.
In above-mentioned detection method, the protein solution that adds the optimum mark amount in aurosol by volume number percent is 2%～3%, stirs 2～3 minutes.
In above-mentioned detection method, the PEG20000 solution of adding is 4.8%～5.2% of cumulative volume, and its concentration is 1%.
In above-mentioned detection method, precipitation is suspended in certain volume to be contained in the damping fluid of 0.2～0.3mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 10000g～100000g, recover with same damping fluid again, concentration is advisable with A1cm/540nm=1.5, anticorrosion with 0.3～0.6mg/ml Sodium azide, put 4 ℃ of preservations.
The present invention adopts immune competition law to detect and compares with the detection of prior art, and mainly contain following advantage: (1) is highly sensitive, and the prior art detection limit is mg, the ug level, and adopt immune competition law check and analysis to be generally ng, pg, even can reach the ag level; (2) high specificity can be told material closely similar on the chemical constitution, even can discern spatial structure; (3) easy and simple to handle, it is few that immune competition law detects required varieties of reagent, and the application of sample program is simple, and the reaction time is short, and measurement and data processing easily are automated.
Embodiment
Embodiment 1
The pre-treatment of honey sample: get 1g honey, put into centrifuge tube, add the 2mL dissolved in distilled water; Add 2mL ethyl acetate vibration 10min; The centrifugal 10min of room temperature 3000g; Pipette 1mL upper strata ethyl acetate (being equivalent to the 0.5g sample) to another test tube, 60 ℃ of following N2 dry up; Residue dissolves with 0.5mL damping fluid 1; Getting 50uL analyzes.
The pre-treatment of shrimp sample: shrimp sample decapsidate, with the equal quality sample of homogenizer; Get the sample that the 3g homogeneous is crossed, put into centrifuge tube, add 6mL ethyl acetate vibration 10min; The centrifugal 10min of room temperature 3000g; Taking out 60 ℃ of following N2 of 2mL upper strata ethyl acetate (being equivalent to the 1g sample) dries up.
The preparation of cleansing solution: Tris54g, boric acid 27.5g with a small amount of DD-H2O dissolving, adds 20ml 0.5mol/L EDTA-Na2 (PH 8.0), adds DD-H2O and is settled to 1000ml.Add 200ml saturated (NH4) 2SO4 solution, 40ml Tween-20 and 480ml DD-H2O, mixing.
The preparation of reagent biotinylated antibody: get biotin 1g, be suspended in 12mlN, N dimethyl formamide DMF, the dicyclohexyl charcoal diimine DCC of adding 0.6g N-maloyl imines HOSU and 0.8g places closed container, and the effect of room temperature magnetic agitation is spent the night.Filtered fluid passed through the rotation evaporate to dryness.Add the washing of 10ml ether, add the crystallization of 200ml isopropyl alcohol then, obtain white powder activation biotin crystal.With DMF BNHS is made into 0.8mg/mL solution; With antibody 0.08mol/L to be marked, pH value is that 8.2 sodium bicarbonate solution is diluted to 1mg/ml, and after the dialysis, with 1mg/ml BNHS: the ratio of antibody to be marked=1: 8 is mixed jolting frequently 1 hour.Put and add 0.01mol/L in the bag filter, pH7.0PBS solution, 4 ℃ of dialysed overnight are diluted to working concentration during use.
Reagent Avidin Preparation of Colloidal Gold: 1. the pH with 0.08mol/L sal tartari adjusting 1ml colloidal gold solution is 8.5; 2. after protein storage liquid to be marked being made serial dilution, get 0.1ml respectively and (contain protein 5～40ug) and be added in the 1ml colloidal gold solution, stirred 1 minute; 3. the concentration that adds (number percent by volume) 4.5% is 0.8%PEG20000 solution; 4. be in the ultracentrifuge of 10000g centrifugal 60 minutes at rotating speed, inhale and remove supernatant; 5. precipitation is suspended in the damping fluid that contains 0.2mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 10000g, recovers with same damping fluid again, anticorrosion with the 0.2mg/ml Sodium azide, put 4 ℃ of preservations.Establish one during use in addition and manage the control tube that does not add protein, add 0.1ml 10%NaCl solution after 5 minutes, left standstill behind the mixing 2 hours, coagulation will take place in unsettled aurosol, can make collaurum the suitableeest stable protein content add 10% again and be the optimum mark protein content.
Getting antibody concentration is 5.94mg/ml, and with the antibody biotinylation, the biotinylated antibody original content is 1.67mg/ml, uses the PBS dilution to 0.02mg/ml,
Spend incubation 20min in 30 after getting 100ul biotinylated antibody (0.02mg/ml) and the abundant mixing of 500ul testing sample, adding 4 of cleansing solutions is coated with on the solid phase carrier that multiple veterinary drug coupled antigen arranges according to little array in above-mentioned, the colloidal gold solution 500ml that adds the Avidin mark, fully after the reaction, add six of cleansing solutions, by reading the result on the scanner, wherein the conversion relation of gray-scale value and residue of veterinary drug amount sees Table 3
Table 3: the conversion relation of gray-scale value and residue of veterinary drug amount
More than be the conversion relation of the inventor's homemade gray-scale value and residue of veterinary drug amount by studying for a long period of time, wherein very nearly the same with the antibiotic content of titer that prior art provides by the concentration (ng/ml) of mass spectrophotometry microbiotic titer.
Embodiment 2:
The pre-treatment of sample and the preparation of cleansing solution are with embodiment 1
The preparation of reagent biotinylated antibody: get biotin 1g, be suspended in 12ml N, N dimethyl formamide DMF, the dicyclohexyl charcoal diimine DCC of adding 0.6g N-maloyl imines HOSU and 0.8g places closed container, and the effect of room temperature magnetic agitation is spent the night.Filtered fluid is through the rotation evaporate to dryness.Add the washing of 10ml ether, add the crystallization of 200ml isopropyl alcohol then, obtain white powder activation biotin crystal.With DMF BNHS is made into 1mg/mL solution; With antibody 0.10mol/L to be marked, pH value is that 9.0 sodium bicarbonate solution is diluted to 2mg/ml, and after the dialysis, with 1mg/ml BNHS: the ratio of antibody to be marked=1: 10 is mixed jolting frequently 3 hours.Put and add 0.05mol/L in the bag filter, pH7.4PBS solution, 4 ℃ of dialysed overnight are diluted to working concentration during use.
Reagent Avidin Preparation of Colloidal Gold: 1. the pH that regulates the 1ml colloidal gold solution with 0.10mol/L sal tartari be 9.0 2. protein storage liquid to be marked is made serial dilution after, get respectively 0.2ml (contain protein 5～40ug) be added to stir in the 1ml colloidal gold solution 2 minutes 3. the concentration of adding (number percent by volume) 5.0% be 1%PEG20000 solution.4. be in the ultracentrifuge of 50000g centrifugal 45 minutes at rotating speed, inhale and remove supernatant.5. precipitation is suspended in the damping fluid that contains 0.4mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 50000g, recovers with same damping fluid again, anticorrosion with the 0.4mg/ml Sodium azide, put 4 ℃ of preservations.Establish one during use in addition and manage the control tube that does not add protein, add 0.1ml 10%NaCl solution after 5 minutes, left standstill behind the mixing 2 hours, coagulation will take place in unsettled aurosol, can make collaurum the suitableeest stable protein content add 10% again and be the optimum mark protein content.
Getting antibody concentration is 6.54mg/ml, and with the antibody biotinylation, the biotinylated antibody original content is 1.76mg/ml, uses the PBS dilution to 0.04mg/ml,
Spend incubation 10min in 37 after getting 100ul biotinylated antibody (0.02mg/ml) and the abundant mixing of 600ul testing sample, adding 4 of cleansing solutions is coated with on the solid phase carrier that multiple veterinary drug coupled antigen arranges according to little array in above-mentioned, the colloidal gold solution 500ml that adds the Avidin mark, fully after the reaction, add six of cleansing solutions, by reading the result on the scanner.
Embodiment 3
The pre-treatment of sample and the preparation of cleansing solution are with embodiment 1
The preparation of reagent biotinylated antibody: get biotin 1g, be suspended in 12mlN, N dimethyl formamide DMF, the dicyclohexyl charcoal diimine DCC of adding 0.6g N-maloyl imines HOSU and 0.8g places closed container, and the effect of room temperature magnetic agitation is spent the night.Filtered fluid passed through the rotation evaporate to dryness.Add the washing of 10ml ether, add the crystallization of 200ml isopropyl alcohol then, obtain white powder activation biotin crystal.With DMF BNHS is made into 1.2mg/mL solution; With antibody 0.12mol/L to be marked, pH value is that 9.2 sodium bicarbonate solution is diluted to 32mg/ml, and after the dialysis, with 1mg/ml BNHS: the ratio of antibody to be marked=1: 15 is mixed jolting frequently 5 hours.Put and add 0.1mol/L in the bag filter, pH7.6PBS solution, 4 ℃ of dialysed overnight are diluted to working concentration during use.
Reagent Avidin Preparation of Colloidal Gold: 1. the pH with 0.12mol/L sal tartari adjusting 1ml colloidal gold solution is 9.5; 2. after protein storage liquid to be marked being made serial dilution, (containing protein 5～40ug) is added in the 1ml colloidal gold solution and stirred 4 minutes to get 0.4ml respectively; 3. the concentration that adds (number percent by volume) 5.5% is 1.2%PEG20000 solution; 4. be in the ultracentrifuge of 100000g centrifugal 30 minutes at rotating speed, inhale and remove supernatant; 5. precipitation is suspended in the damping fluid that contains 0.5mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 100000g, recovers with same damping fluid again, anticorrosion with the 0.8mg/ml Sodium azide, put 4 ℃ of preservations.Establish one during use in addition and manage the control tube that does not add protein, add 0.1ml 10%NaCl solution after 5 minutes, left standstill behind the mixing 2 hours, coagulation will take place in unsettled aurosol, can make collaurum the suitableeest stable protein content add 10% again and be the optimum mark protein content.
Getting antibody concentration is 6.74mg/ml, and with the antibody biotinylation, the biotinylated antibody original content is 1.78mg/ml, uses the PBS dilution to 0.03mg/ml,
Spend incubation 5min in 38 after getting 200ul biotinylated antibody (0.02mg/ml) and the abundant mixing of 500ul testing sample, adding 4 of cleansing solutions is coated with on the solid phase carrier that multiple veterinary drug coupled antigen arranges according to little array in above-mentioned, the colloidal gold solution 500ml that adds the Avidin mark, fully after the reaction, add six of cleansing solutions, by reading the result on the scanner.
Specific embodiment described herein 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 claims.
Claims (9)
1, the detection method of a kind of fast detecting food, agricultural byproducts veterinary drug residue thing may further comprise the steps:
(1), multiple veterinary drug coupled antigen is wrapped quilt according to microarray point on solid phase carrier and to it; Wherein fixing veterinary drug antigen on the solid-phase matrix;
(2), will mix through the biotinylated antibody of diluted and testing sample, with the immune response that is at war with of multiple veterinary drug envelope antigen fixing on the solid phase carrier, cleansing solution washing; If testing sample does not contain veterinary drug in the course of reaction, the then multiple veterinary drug envelope antigen combination of fixing on antibody and the solid phase carrier; If testing sample contains veterinary drug, then biotin labeled antibody can combine with the veterinary drug in the testing sample, and by wash-out, described is 2: 5～1: 6 through the biotinylated antibody of diluted and the blending ratio of sample;
(3), the collaurum that adds the Avidin mark amplifies and dye, by scanner acquisition gray scale result, wherein said Avidin labeling method is:
1. the pH with 0.08～0.12mol/L carbonate adjusting aurosol is 8.5～9.5;
2. add in aurosol by volume that number percent is 1%～4% protein solution, stirred 1～4 minute, protein content is 5～40 μ g in the wherein said protein solution;
3. adding number percent by volume and be 4.5%～5.5% concentration is 0.8～1.2%PEG20000 solution;
4. be in the ultracentrifuge of 10000g～100000g centrifugal 30～60 minutes in rotating speed, inhale and remove supernatant;
5. precipitation is suspended in the damping fluid that contains 0.2～0.5mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 10000g～100000g, recover with same damping fluid, concentration is with A1cm/540nm=1.5 again, anticorrosion with 0.2～0.8mg/ml Sodium azide, put 4 ℃ of preservations.
2, the detection method of a kind of fast detecting food according to claim 1, agricultural byproducts veterinary drug residue thing, it is characterized in that, the biotinylated antibody of process diluted mixes the post-reacted time with sample be 5～20min, and the temperature of reaction is 30 ℃～38 ℃.
3, the detection method of a kind of fast detecting food according to claim 1 and 2, agricultural byproducts veterinary drug residue thing, it is characterized in that, the labeling method of the biotinylated antibody of described process diluted is: with DMF BNHS is made into 0.8～1.2mg/mL solution, with 0.08～0.12mol/L, pH8.2～9.2NaHCO
3With antibody purified dilution is 1～3mg/mL, is to mix in 1: 8～1: 15 by the volumetric ratio of BNHS: IgG, reacts 1～5h under the stirring at room, the bag filter of packing into, adding 0.01mol/L～1mol/L, pH7.0～7.6PBS solution, 4 ℃ of dialysed overnight.
4, the detection method of a kind of fast detecting food according to claim 3, agricultural byproducts veterinary drug residue thing, it is characterized in that, the labeling method of described biotinylated antibody is: with DMF BNHS is made into 1mg/mL solution, uses 0.1mol/L, the NaHCO of pH9.0
3Antibody purified dilution is 2mg/mL, is 1: 10 by the volumetric ratio of BNHS: IgG, reacts 2～4h under the stirring at room, the bag filter of packing into, adding 0.05mol/L, pH7.2PBS solution, 4 ℃ of dialysed overnight.
5, the detection method of a kind of fast detecting food according to claim 1, agricultural byproducts veterinary drug residue thing is characterized in that, carbonate is sal tartari in the described Avidin labeling method step 1.
6, the detection method of a kind of fast detecting food according to claim 5, agricultural byproducts veterinary drug residue thing is characterized in that, described Avidin labeling method step 1 is: with 0.08mol/L～0.12mol/L K
2CO
3Regulate aurosol pH to 8.8～9.2.
7, the detection method of a kind of fast detecting food according to claim 1, agricultural byproducts veterinary drug residue thing is characterized in that, the protein solution that adds labelled amount in aurosol by volume number percent is 2%～3%, stirs 2～3 minutes.
8, the detection method of a kind of fast detecting food according to claim 1, agricultural byproducts veterinary drug residue thing is characterized in that, the PEG20000 solution of adding is 4.8%～5.2% of cumulative volume, and its concentration is 1%.
9, the detection method of a kind of fast detecting food according to claim 1, agricultural byproducts veterinary drug residue thing, it is characterized in that, precipitation is suspended in certain volume to be contained in the damping fluid of 0.2～0.3mg/ml PEG20000, after rotating speed is the ultracentrifuge centrifugation of 10000g～100000g, recover with same damping fluid again, anticorrosion with 0.3～0.6mg/ml Sodium azide, put 4 ℃ of preservations.
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| CN106370844A (en) * | 2016-08-30 | 2017-02-01 | 吕炜锋 | Dual colloidal gold test strip for detecting human bone metabolic biomarker and detection device applying same |
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