CN202383142U - Colloidal gold test paper for detecting abamectin (AVM) residual - Google Patents
Colloidal gold test paper for detecting abamectin (AVM) residual Download PDFInfo
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- CN202383142U CN202383142U CN2011204507750U CN201120450775U CN202383142U CN 202383142 U CN202383142 U CN 202383142U CN 2011204507750 U CN2011204507750 U CN 2011204507750U CN 201120450775 U CN201120450775 U CN 201120450775U CN 202383142 U CN202383142 U CN 202383142U
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Abstract
The utility model relates to a piece of colloidal gold test paper for detecting abamectin (AVM) residual, which comprises a plastic substrate, a sample loading region, a colloidal-gold labeling region, a color developing region and a water absorption region. Due to adoption of a competition law principle, the AVM in a sample competes with a colloidal-gold labeled AVM envelope antigen (AVM-OVA) and is further combined with an AVM monoclonal antibody on a detection strip to realize color development and establish a corresponding relationship between the AVM content in the sample and the quantity of color developing strips of the detection strip, and furthermore, the AVM content in the sample is judged according to the strip color-developing quantity in the color developing region. The colloidal gold test paper disclosed by the utility model has the characteristics of simple operation, sensitivity and specificity, convenience for carrying and the like and can be applied to AVM residual detection of animal-derived foods and agricultural fruit and vegetable products.
Description
Technical field
The utility model relates to Detecting Pesticide test strips in animal-derived food and the fruits and vegetables agricultural products, relates to a kind of residual colloid gold test paper of AVM that detects specifically.
Background technology
(Avermectin AVM) is a kind of macrolide antibiotic class Insecticidal and acaricidal agent that was obtained from the tunning of streptomycete MA-4680 in 1976 by Japanese scientist big village intelligence and U.S. Merck company to AVM.AVM belongs to the insect never poison, mainly disturbs the insect nervous physiology movable, stimulates to discharge C
2Aminobutyric suppresses nerve conduction, and the insect paralysis is poisoned and dead, and tool is tagged and stomach poison function, and absorption in not having is present most widely used anti-parasite medicine, is widely used in the healthy aquaculture of disease of agricultural plants control and livestock and poultry.
Though AVM belongs to microbial pesticide, it has extremely strong fat-soluble, no matter with which kind of administration all can be absorbed well, can be distributed widely in body tissue, longer duration in the body, thereby eliminate slowly.The World Health Organization (WHO) regards as height with AVM poisons compound.Research shows that AVM is to the LD of rat
50Be 10 mg/kg, belong to high-toxic pesticide.Research finds that also AVM has potential toxicity to mammiferous fertility, when the experimental mouse intake is 1.19-2.13 mg/! Zhi ﹒ d) after, the fertility fecundity of male wister rat significantly descends, and the probability that stillborn foetus takes place the spouse obviously improves.Therefore, the MRL MRL (Maximum Residue Limits) to AVM requires very strictness in the world, and the detection of its residual index is attached great importance to, and has formulated quite strict MRL value.The Ministry of Agriculture stipulates that its MRL in citrus is 20 μ g/kg, is 50 μ g/kg in the leaf vegetables; European Union stipulates that its MRL in agricultural product such as fruit and vegetable food, cereal is 10 μ g/kg; Korea S stipulates that its MRL in apple is 20 μ g/kg, and celery is 50 μ g/kg; Israel stipulates that its MRL in fruits and vegetables agricultural product such as cucumber, eggplant, peach, strawberry is 10 μ g/kg; The MRL that Australia is defined in the products such as apple, pears, tomato is 10 μ g/kg.
At present, the method for AVM residue detection mainly contains thin-layered chromatography (TLC), vapor-phase chromatography (GC); High performance liquid chromatography-uv detection method (HPLC-UV); High performance liquid chromatography-fluorescence detection (HPLC-FLD), gas-matter coupling method (HPLC-MS), liquid-matter coupling method (LC-MS); Capillary electrophoresis (CE), ELISA (ELISA) etc.These methods exist the instrument and equipment complicacy, and operation steps is many, and process is loaded down with trivial details, and testing cost is high, need carry out in the laboratory, and require special problems such as technician.
Summary of the invention
The purpose of the utility model is to provide
A kind of residual colloid gold test paper of AVM that detects, it hasHigh specificity, highly sensitive, accurate, convenient, cheap, the popular residual colloid gold test paper of detection AVM.
A kind of residual colloid gold test paper of AVM that detects of the utility model is by plastic base, sample application zone, gold-marking region, reaction solution district and suction zones, and the above-mentioned overlapping successively overlap joint of belt edge of respectively distinguishing is pasted and fixed on the plastic base; In the reaction solution district, be provided with three and detect band and a quality control band.
As the further improvement of the utility model, described gold-marking region is made up of gold mark AVM envelope antigen AVM-OVA.
As the further improvement of the utility model, described AVM envelope antigen AVM-OVA is formed by AVM and protein carrier chicken egg white OVA coupling.
As the further improvement of the utility model, described gold mark AVM envelope antigen AVM-OVA is formed by colloidal nano gold grain mark AVM envelope antigen AVM-OVA, and its concentration is 5 μ g/L ~ 15 μ g/L.
As the further improvement of the utility model, described three are detected the AVM monoclonal antibody that band is coated with the variable concentrations gradient.
As the further improvement of the utility model, described three to detect band be respectively that to encapsulate concentration be that first of 1.0 μ g/L detect band, and encapsulating concentration is that second of 2.0 μ g/L detect band, and encapsulating concentration is that the 3rd of 5.0 μ g/L detect band.
As the further improvement of the utility model, a described quality control band is coated with certain density mouse-anti chicken egg white antibody.
As the further improvement of the utility model, described suction zones is made up of filter paper.
When this test strips was used for test sample, the sample application zone of pretreated sample drop in this test strips, sample at first passed through gold-marking region because capillary action is carried out lateral chromatography, and sample can be marked the AVM envelope antigen with gold and redissolve, and carries out lateral chromatography then together.When through the color developing detection district on the nitrocellulose filter, gold mark AVM envelope antigen meeting and detect with on AVM monoclonal antibody and the mouse-anti chicken egg white antibody on the quality control band carry out the antigen-antibody idiosyncrasy and develop the color.Surpass finite concentration (1.0 μ g/L if contain AVM or its content in the sample; 2.0 μ g/L; 5.0 in the time of μ g/L), its just together with the competition of gold mark AVM envelope antigen with detection with on the AVM monoclonal antibody that encapsulates combines since in the sample AVM compare molecular weight with gold mark AVM envelope antigen little a lot; Chromatography time space steric hindrance is also very little; Thereby make that AVM combines with the AVM monoclonal antibody than gold mark AVM comlete antigen sooner more easily in the sample, thus make the AVM envelope antigen can not with detect band AVM monoclonal antibody and combine colour developing, the golden like this AVM of marking envelope antigen can continue chromatography and all the other AVM monoclonal antibodies and anti-chicken egg white antibodies and develop the color.
The beneficial effect of the utility model: improved the specificity, the sensitivity that detect.Utilize colloidal nano gold grain mark AVM comlete antigen (envelope antigen AVM-OVA); It is visual to have improved its testing result, has further improved sensitivity, has broken away from the constraint of particular instrument; Reduced cost, made that the use object of this test strips is accurate, convenient, cheap, popular more.
Description of drawings
Fig. 1 is a kind of structural representation that detects the colloidal gold strip of AVM residual content;
Fig. 2 is the exploded view of Fig. 1.
Fig. 3 is the testing result key diagram.
Embodiment
A kind of residual colloid gold test paper of AVM that detects of the utility model is characterized in that plastic base 1, sample application zone 2, gold-marking region 3, reaction solution district 4 and suction zones 9, and the above-mentioned overlapping successively overlap joint of belt edge of respectively distinguishing is pasted and fixed on the plastic base 1; Reaction solution district 4 is provided with three and detects band and a quality control band 8.Described gold-marking region 3 is made up of gold mark AVM envelope antigen AVM-OVA; Article three, detect and be with its concentration of AVM monoclonal antibody that is coated with the variable concentrations gradient to be respectively: the concentration that encapsulates that wherein detects with 5 is 1.0 μ g/L, and the concentration that encapsulates that detects with 6 is 2.0 μ g/L, and the concentration that encapsulates that detects with 7 is 5.0 μ g/L; Quality control band 8 is coated with certain density mouse-anti chicken egg white antibody; Suction zones 9 is made up of filter paper.
Described AVM envelope antigen AVM-OVA is formed by AVM and protein carrier chicken egg white OVA coupling.
Described AVM envelope antigen AVM-OVA is formed by colloidal nano gold grain mark AVM envelope antigen AVM-OVA, and its concentration is 5 μ g/L ~ 15 μ g/L.
A kind of preparation that detects the residual colloid gold test paper of AVM of the utility model:
1, AVM MONOCLONAL ANTIBODIES SPECIFIC FOR:
(1) AVM is become carboxyl AVM with the succinic acid carboxylation, with carbodlimide method it is joined respectively with hemocyanin KLH, chicken egg white OVA lotus root again, join albumen, identify that with the UV scanning method its lotus root joins effect with dialysis purifying AVM lotus root.Finally process AVM comlete antigen (immunizing antigen AVM-KLH, envelope antigen AVM-OVA);
(2) with AVM immunizing antigen AVM-KLH immune balb/c mice, after three immunity, survey the mice serum antibody titer, carry out booster immunization at last, getting the high mouse of tiring is the splenocyte supplier;
(3) with the assorted azaguanine domestication of 8-SP2/0 cell, treat that its domestication is good after, merge according to the mouse boosting cell of the classical zooblast fusion method antibody horizontal that generation in SP2/0 cell and the body is high;
(4) with AVM envelope antigen AVM-OVA coated elisa plate, indirect elisa method detects the fused cell that can secrete AVM antibody, and carries out subclone according to limiting dilution assay, up to filtering out the stable fusion cell line that can successfully secrete the AVM monoclonal antibody;
Enlarged culture is carried out in the stable positive cell strain that can secrete the AVM monoclonal antibody that (5) will filter out, and it is injected into the pure lines BALB/C mice intraperitoneal of taming through paraffin in advance, makes it produce the ascites that contains secretion AVM monoclonal antibody in a large number;
(6) ascites of collecting is concentrated, purifies, and then obtain the AVM monoclonal antibody.
2, the preparation of mouse-anti chicken egg white (OVA) antibody:
(1) with chicken egg white according to routine immunization program immunity BALB/C mice, three immunity back ELISA methods are measured mouse antibodies and are tired, to confirm the mouse antibodies level;
(2) the highest mouse of will tiring carries out eyeball excise method blood sampling, separate its serum, obtain mouse-anti chicken egg white OVA antibody according to sad-ammonium sulfate method purifying.
3, the preparation of colloidal nano gold grain:
(1) 0.01% of adding a certain amount of (100 mL) aqueous solution of chloraurate in the beaker of cleaning is heated to boiling then;
(2) trisodium citrate aqueous solution 1 mL of adding finite concentration (1.0%) under stirring continues to boil 10 min, and the reaction system color gradually becomes aubergine from golden yellow;
(3) leave standstill cooling after, return to original volume with deionized water, finally process qualified (particle diameter is about 40 nm) colloidal nano gold grain.
4, colloid gold label AVM envelope antigen AVM-OVA:
(l) confirm optimal pH and the concentration of colloid gold label AVM envelope antigen AVM-OVA through preliminary experiment;
(2) with the colloidal gold solution that makes through be adjusted to optimal pH (pH 8.5) with 0.1 M sal tartari;
(3) with processing such as the further dialysis of AVM envelope antigen AVM-OVA process, filtrations, remove impurity such as some ions that influence golden mark process and particle as far as possible;
(4) under the electromagnetic agitation condition, treated AVM envelope antigen AVM-OVA is dropwise added in the colloidal gold solution of 100 mL according to optimizing the condition concentration (3.0 mL) that;
Behind (5) 10 min, add 10% BSA to its concentration be 1%, to stablize its reaction system, continue to stir;
The gold mark AVM envelope antigen AVM-OVA solution that (6) will make is through centrifugal (15000 g, 4
oC, 30 min) after, carefully draw supernatant, with the PBS that contains l% BSA recover original volume resuspended after, centrifugal again, promptly repeated washing is 3 times;
(7) will precipitate final resuspended to suitable concn with the PBS that contains stabilizing agent (1% BSA or 0.3 mg/mL PEG).Adding a certain amount of 0. 5 mg/mL Sodium azides can be long-term by 4
oC preserves.
5, the assembling of the residual test paper strip for semi-quantitative detecting of AVM:
(1) (pH 7.4 spun glass to be passed through the PBS that contains l% BSA and 1% Tween-20; 0.01 M) after the activation processing; Be immersed in 30 min in the gold mark AVM envelope antigen AVM-OVA solution, natural air drying can be marked AVM envelope antigen AVM-OVA with gold and be coated on the glass fibre membrane;
(2) spray the AVM monoclonal anti liquid solution band of three gradient concentrations successively as detecting band at interval 0.2 cm on the nitrocellulose filter, its concentration is followed successively by 1.0 μ g/L, 2.0 μ g/L, 5.0 μ g/L.Mouse-anti chicken egg white OVA antibody is quality control band.Then nitrocellulose filter is sealed 2 h, natural air drying with the PBS (pH 7.4,0.01 M) that contains l% BSA;
(3) last; With application of sample film (principal ingredient is filter paper or spun glass, as sample region), be coated with gold mark AVM envelope antigen glass fibre membrane (gold-marking region), encapsulate the nitrocellulose filter (colour developing district), the absorbent filter (suction zones) that detect band and quality control band and be attached to successively and process AVM residue detection test paper on the plastic bottom board.
A kind of use that detects the residual colloid gold test paper of AVM of the utility model:
(1) testing sample is carried out pre-service
Liquid sample comprises animal body fluid (serum, urine, milk etc.) and liquid beverage (milk, fruit juice etc.), need not carry out pre-service, and perhaps waiting doubly with distilled water, dilution gets final product.
The meat sample is got 1.0 g samples and is put to smash to pieces in the cup and mix with 10. 0 mL acetone solvents, smashs 2 min to pieces at a high speed, and supersonic oscillations 30 min are with centrifugal 10 min of 10 000 rpm.Get supernatant 1mL, subsequent use.
The fruits and vegetables sample is got fruits and vegetables sample 10.0 g after the pulverizing, adds 50 mL acetonitriles; In the even at a high speed matter device of 14 000rpm, behind even matter 2 min, use filter paper filtering, filtrating is collected in the 100 mL tool plug graduated cylinders that 5-7 g sodium chloride is housed; Collect filtrating, cover stopper, concuss 2 min; At room temperature static 30 min make acetonitrile phase and water layering.Draw 10 mL solution from acetonitrile mutually, subsequent use.
(2) micro-sample is splashed into sample application zone, or test strips stretched into to begin in the sample to detect.Get final product observations behind the 5-10 min.
(3) result judges (combining Figure of description 3)
All develop the color if three detect band and quality control band, then interpret sample is negative, and does not contain AVM or AVM content in the sample and is lower than 1.0 μ g/L, shown among Fig. 31; Be with 5 not develop the color if detect, and other two detections are with and the quality control band colour developing, then interpret sample is positive, and AVM content is between 1.0 μ g/L-2.0 μ g/L, shown among Fig. 32 in the sample; Be with 5 to be with 6 all not develop the color if detect with detection, detect be with 7 with the quality control band colour developing, then interpret sample is positive, AVM content is between 2.0 μ g/L-5.0 μ g/L, shown among Fig. 33 in the sample; Be with 5,6,7 not develop the color if detect, have only the quality control band colour developing, then interpret sample is positive, and AVM content meets or exceeds 5.0 μ g/L in the sample, shown among Fig. 34; All do not develop the color if detect band, quality control band does not develop the color yet, and explains that then this test strips lost efficacy, and can not use, and detects again after the test strips that must more renew, shown among Fig. 35.
Claims (2)
1. one kind is detected the residual colloid gold test paper of AVM; It is characterized in that plastic base (1), sample application zone (2), gold-marking region (3), reaction solution district (4) and suction zones (9); The above-mentioned overlapping successively overlap joint of belt edge of respectively distinguishing is pasted and fixed on the plastic base (1); On reaction solution district (4), be provided with three and detect a band and a quality control band (8).
2. a kind of residual colloid gold test paper of AVM that detects described in claim 1 is characterized in that suction zones (9) is made up of filter paper.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011204507750U CN202383142U (en) | 2011-11-15 | 2011-11-15 | Colloidal gold test paper for detecting abamectin (AVM) residual |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011204507750U CN202383142U (en) | 2011-11-15 | 2011-11-15 | Colloidal gold test paper for detecting abamectin (AVM) residual |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407146A (en) * | 2014-12-02 | 2015-03-11 | 河南省科学院生物研究所有限责任公司 | Preparation method of abamectin colloidal gold test strip |
| CN104807815A (en) * | 2015-04-30 | 2015-07-29 | 周二春 | PH (Potential of Hydrogen) test paper card |
| CN111751536A (en) * | 2020-07-16 | 2020-10-09 | 生态环境部南京环境科学研究所 | Device and method for rapidly detecting residual quantity of abamectin B2a in soil |
-
2011
- 2011-11-15 CN CN2011204507750U patent/CN202383142U/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407146A (en) * | 2014-12-02 | 2015-03-11 | 河南省科学院生物研究所有限责任公司 | Preparation method of abamectin colloidal gold test strip |
| CN104807815A (en) * | 2015-04-30 | 2015-07-29 | 周二春 | PH (Potential of Hydrogen) test paper card |
| CN111751536A (en) * | 2020-07-16 | 2020-10-09 | 生态环境部南京环境科学研究所 | Device and method for rapidly detecting residual quantity of abamectin B2a in soil |
| CN111751536B (en) * | 2020-07-16 | 2022-04-19 | 生态环境部南京环境科学研究所 | Device and method for rapidly detecting residual quantity of abamectin B2a in soil |
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Granted publication date: 20120815 Termination date: 20121115 |