CN202383142U - Colloidal gold test paper for detecting abamectin (AVM) residual - Google Patents

Abstract

The utility model relates to a colloidal gold test paper for detecting residual abamectin, which is composed of a plastic bottom plate, a sample adding area, a gold label area, a color developing area and a water absorbing area. The principle of competition method is used to make the AVM in the sample compete with the gold-labeled AVM-coated antigen AVM-OVA, so as to combine with the AVM monoclonal antibody on the detection belt to develop color, and establish the relationship between the AVM content in the sample and the number of color strips in the detection belt. The relationship, and then judge the AVM content in the sample according to the number of colored bands in the colored area. The utility model has the characteristics of simple operation, sensitive and specific, convenient to carry and the like. It can be applied to the detection of AVM residues in animal source foods and agricultural products of fruits and vegetables.

Description

A kind of colloidal gold test paper that detects abamectin residue

technical field

The utility model relates to a test paper strip for detecting pesticide residues in animal source foods and agricultural products of fruits and vegetables, in particular to a colloidal gold test paper for detecting abamectin residues.

Background technique

Avermectin (Avermectin, AVM) is a macrolide antibiotic insecticide and acaricide obtained from the fermentation product of Streptomyces MA-4680 by Japanese scientist Omura Satoshi and American Merck Company in 1976. AVM is an insect nerve agent, which mainly interferes with the neurophysiological activities of pests, stimulates the release of _C2 aminobutyric acid, inhibits nerve conduction, and paralyzes and poisons the pests to death. It has contact and stomach poisoning effects, and has no systemic properties. It is currently the most widely used Antiparasitic drugs are widely used in the prevention and control of crop diseases and the healthy breeding of livestock and poultry.

Although AVM is a microbial-derived pesticide, it is highly fat-soluble and can be well absorbed regardless of the route of administration. It can be widely distributed in the tissues of the body and lasts for a long time in the body, so its elimination is slow. The World Health Organization recognizes AVM as a highly toxic compound. Studies have shown that the LD ₅₀ of AVM to rats is 10 mg/kg, which is a highly toxic pesticide. The study also found that AVM has potential toxicity to the reproductive ability of mammals. When the intake of experimental mice is 1.19-2.13 mg/(only.d), the reproductive ability of male mice is significantly reduced, and the probability of stillbirth in the spouse Significantly improved. Therefore, the international requirements for the MRL (Maximum Residue Limits) of AVM are very strict, and the detection of its residue indicators is very important, and a very strict MRL value has been formulated. The Chinese Ministry of Agriculture stipulates that its MRL in citrus is 20 μg/kg, and that in leafy vegetables is 50 μg/kg; the European Union stipulates that its MRL in fruit and vegetable products, grains and other agricultural products is 10 μg/kg; South Korea stipulates that its MRL in apples 20 μg/kg for celery, 50 μg/kg for celery; Israel stipulates that the MRL for cucumber, eggplant, peach, strawberry and other fruit and vegetable agricultural products is 10 μg/kg; Australia stipulates that the MRL for apples, pears, tomatoes and other products is 10 μg/kg.

At present, the methods for AVM residue detection mainly include thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography-ultraviolet detection (HPLC-UV), high-performance liquid chromatography-fluorescence detection (HPLC-FLD). ), gas-mass spectrometry (HPLC-MS), liquid-mass spectrometry (LC-MS), capillary electrophoresis (CE), enzyme-linked immunoassay (ELISA), etc. These methods have the problems of complex instruments and equipment, many operation steps, cumbersome process, high detection cost, need to be carried out in a laboratory, and require specialized technical personnel.

Contents of the invention

The purpose of this utility model is to provide a kind of colloidal gold test paper that detects abamectin residue, it has strong specificity, high sensitivity, accurate, convenient, cheap, the colloidal gold test paper that detects abamectin residue of popularity.

A colloidal gold test paper for detecting residual abamectin of the present utility model is composed of a plastic substrate, a sample adding area, a gold label area, a reaction color development area and a water absorption area. On the plastic substrate; three detection bands and one quality control band are arranged on the reaction color development area.

As a further improvement of the present invention, the gold-labeled area is composed of gold-labeled AVM-coated antigen AVM-OVA.

As a further improvement of the present invention, the AVM coating antigen AVM-OVA is formed by coupling AVM and a protein carrier chicken ovalbumin OVA.

As a further improvement of the present invention, the gold-labeled AVM-coated antigen AVM-OVA is formed by colloidal gold nanoparticles labeled AVM-coated antigen AVM-OVA, and its concentration is 5 μg/L-15 μg/L.

As a further improvement of the utility model, the three detection zones are coated with AVM monoclonal antibodies with different concentration gradients.

As a further improvement of the present utility model, the three detection zones are respectively the first detection zone with a coating concentration of 1.0 μg/L, the second detection zone with a coating concentration of 2.0 μg/L, and a coating concentration of 5.0 μg /L for the third detection zone.

As a further improvement of the utility model, the one quality control belt is coated with a certain concentration of mouse anti-chicken ovalbumin antibody.

As a further improvement of the utility model, the water-absorbing area is made of filter paper.

When the test strip is used to detect samples, the pretreated sample is dropped on the sample loading area of the test strip, and the sample undergoes lateral chromatography due to capillary action, and first passes through the gold label area, and the sample will be gold-labeled AVM The coated antigens were reconstituted and then subjected to lateral flow chromatography together. When passing through the chromogenic detection area on the nitrocellulose filter membrane, the antigen-antibody-specific reaction of the gold-labeled AVM-coated antigen with the AVM monoclonal antibody on the detection strip and the mouse anti-chicken ovalbumin antibody on the quality control strip is obvious. color. If the sample contains AVM or its content exceeds a certain concentration (1.0 μg/L, 2.0 μg/L, 5.0 μg/L), it will compete with the gold-labeled AVM-coated antigen and compete with the AVM monoclonal coated on the detection strip Antibody binding, because the molecular weight of AVM in the sample is much smaller than that of the gold-labeled AVM-coated antigen, and the steric hindrance during chromatography is also small, so that the AVM in the sample is faster and easier to bind to the AVM monoclonal antibody than the gold-labeled AVM complete antigen Combination, thus making the AVM coating antigen unable to combine with the detection band AVM monoclonal antibody for color development, so that the gold-labeled AVM coating antigen will continue to be chromatographically combined with the rest of the AVM monoclonal antibody and anti-chicken ovalbumin antibody for color development.

Beneficial effects of the utility model: the detection specificity and sensitivity are improved. The use of colloidal gold nanoparticles to label the complete antigen of AVM (coated antigen AVM-OVA) improves the visibility of the test results, further improves the sensitivity, gets rid of the shackles of specific instruments, and reduces the cost. The object of use is more accurate, convenient, cheap and popular.

Description of drawings

Fig. 1 is a structural representation of a colloidal gold test strip for detecting residual content of abamectin;

FIG. 2 is an exploded view of FIG. 1 .

Figure 3 is an illustration of the test results.

Detailed ways

A colloidal gold test paper for detecting residual abamectin of the present utility model is characterized in that a plastic substrate 1, a sample adding area 2, a gold label area 3, a reaction color development area 4 and a water absorption area 9, and the edges of the above-mentioned areas overlap in sequence Overlapped, pasted and fixed on the plastic substrate 1; the reaction color development area 4 is provided with three detection bands and a quality control band 8. The gold label area 3 is composed of the gold label AVM coated antigen AVM-OVA; the three detection zones are coated with AVM monoclonal antibodies with different concentration gradients, and the concentrations are respectively: the coating concentration of the detection zone 5 is 1.0 μg/ L, the coating concentration of the detection zone 6 is 2.0 μg/L, the coating concentration of the detection zone 7 is 5.0 μg/L; the quality control zone 8 is coated with a certain concentration of mouse anti-chicken ovalbumin antibody; the water absorption area 9 is composed of filter paper composition.

The AVM coating antigen AVM-OVA is formed by coupling AVM and a protein carrier chicken ovalbumin OVA.

The AVM-coated antigen AVM-OVA is formed by colloidal gold nanoparticles labeled AVM-coated antigen AVM-OVA, and its concentration is 5 μg/L˜15 μg/L.

A kind of preparation of the colloidal gold test paper that detects Abamectin residue of the present utility model:

1. Preparation of AVM monoclonal antibody:

(1) Carboxylate AVM with succinic acid into carboxyl AVM, then couple them with hemocyanin KLH and chicken ovalbumin OVA respectively by carbodiimide method, purify AVM coupling protein by dialysis, and use UV scanning method Identify its coupling effect. Finally, the complete AVM antigen (immune antigen AVM-KLH, coating antigen AVM-OVA) is made;

(2) BALB/C mice were immunized with the AVM immune antigen AVM-KLH. After three immunizations, the serum antibody titer of the mice was measured, and finally a booster immunization was performed, and the mouse with the highest titer was selected as the splenocyte provider;

(3) Domesticate SP2/0 cells with 8-azaguanine, and after they are domesticated, fuse SP2/0 cells with mouse splenocytes that produce high antibody levels in vivo according to the classical animal cell fusion method;

(4) Coat the microtiter plate with AVM-coated antigen AVM-OVA, detect fusion cells capable of secreting AVM antibody by indirect ELISA, and perform subcloning according to the limited dilution method until a stable cell that can successfully secrete AVM monoclonal antibody is screened out. fusion cell line;

(5) Expand the selected stable positive cell line capable of secreting AVM monoclonal antibody, and inject it into the peritoneal cavity of pure-line BALB/C mice that have been acclimated to paraffin in advance, so that they can produce a large amount of AVM-secreting Ascites with monoclonal antibodies;

(6) Concentrate and purify the collected ascites to obtain AVM monoclonal antibody.

2. Preparation of mouse anti-ovalbumin (OVA) antibody:

(1) Immune BALB/C mice with chicken ovalbumin according to the routine immunization procedure, and measure the mouse antibody titer by ELISA after three immunizations to determine the mouse antibody level;

(2) Blood was collected from the mouse with the highest titer by enucleation, its serum was separated, and the mouse anti-ovalbumin OVA antibody was obtained by purification according to the octanoic acid-ammonium sulfate method.

3. Preparation of colloidal gold nanoparticles:

(1) Add a certain amount (100 mL) of 0.01% chloroauric acid aqueous solution into a clean beaker, then heat to boiling;

(2) Add 1 mL of trisodium citrate aqueous solution with a certain concentration (1.0%) under stirring state, and continue to boil for 10 min, the color of the reaction system gradually changes from golden yellow to purple;

(3) After standing and cooling, restore the original volume with deionized water, and finally make qualified colloidal gold nanoparticles (with a particle size of about 40 nm).

4. Colloidal gold-labeled AVM-coated antigen AVM-OVA:

(l) Determine the optimal pH value and concentration of colloidal gold-labeled AVM-coated antigen AVM-OVA through preliminary experiments;

(2) Adjust the prepared colloidal gold solution to the optimum pH value (pH 8.5) with 0.1 M potassium carbonate;

(3) The AVM-coated antigen AVM-OVA was further processed by dialysis and filtration to remove as much as possible impurities such as ions and particles that affect the gold labeling process;

(4) Under the condition of electromagnetic stirring, add the treated AVM-coated antigen AVM-OVA dropwise into 100 mL colloidal gold solution according to the optimized conditional concentration (3.0 mL);

(5) After 10 minutes, add 10% BSA to a concentration of 1% to stabilize the reaction system and continue to stir evenly;

(6) Centrifuge the prepared gold-labeled AVM-coated antigen AVM-OVA solution (15000 g, 4 ^o C, 30 min), carefully aspirate the supernatant, and resuspend with PBS containing 1% BSA to restore the original volume , and then centrifuged, that is, repeated washing 3 times;

(7) Finally, resuspend the pellet with PBS containing stabilizer (1% BSA or 0.3 mg/mL PEG) to an appropriate concentration. A certain amount of 0.5 mg/mL sodium azide can be added for long-term storage at 4 ^o C.

5. Assembly of test strips for semi-quantitative detection of AVM residues:

(1) After the glass fiber was activated by PBS containing 1% BSA and 1% Tween-20 (pH 7.4, 0.01 M), it was immersed in the gold-labeled AVM-coated antigen AVM-OVA solution for 30 min, and air-dried naturally. The gold-labeled AVM-coated antigen AVM-OVA can be coated on the glass fiber membrane;

(2) Spray three bands of AVM monoclonal antibody solution with gradient concentrations at intervals of 0.2 cm on the nitrocellulose filter membrane as detection bands, the concentrations of which are 1.0 μg/L, 2.0 μg/L, and 5.0 μg/L. Mouse anti-chicken ovalbumin OVA antibody was used as a quality control band. Then the nitrocellulose membrane was blocked with PBS containing 1% BSA (pH 7.4, 0.01 M) for 2 h, and air-dried naturally;

(3) Finally, add the sample membrane (the main component is filter paper or glass fiber, as the sampling area), the glass fiber membrane sprayed with gold-labeled AVM-coated antigen (gold label area), the coated detection zone and the quality control zone. Nitrocellulose filter membrane (color developing area) and water-absorbing filter paper (water-absorbing area) are pasted on the plastic bottom plate in turn to make AVM residue detection test paper.

The use of a kind of colloidal gold test paper that detects Abamectin residue of the present utility model:

(1) Pretreat the sample to be tested

Liquid samples, including animal body fluids (serum, urine, milk, etc.) and liquid beverages (milk, juice, etc.), do not need to be pretreated, or can be diluted with distilled water.

For meat samples, take 1.0 g sample and mix it with 10.0 mL of acetone solvent in a mashing cup, mash at high speed for 2 min, ultrasonically oscillate for 30 min, and centrifuge at 10 000 rpm for 10 min. Take 1 mL of the supernatant and set aside.

For fruit and vegetable samples, take 10.0 g of crushed fruit and vegetable samples, add 50 mL of acetonitrile, homogenize in a high-speed homogenizer at 14 000 rpm for 2 min, filter with filter paper, and collect the filtrate into 100 mL of 5-7 g sodium chloride Collect the filtrate in a stoppered measuring cylinder, cover with a stopper, shake vigorously for 2 min, and let stand at room temperature for 30 min to separate the acetonitrile phase and the aqueous phase. Aspirate 10 mL of the solution from the acetonitrile phase and set aside.

(2) Drop a small amount of sample into the sample loading area, or insert the test strip into the sample to start the test. The results can be observed after 5-10 minutes.

(3) Judgment of the results (combined with Figure 3 of the instruction manual)

If the three detection bands and the quality control band are all colored, it means that the sample is negative, and the sample does not contain AVM or the AVM content is lower than 1.0 μg/L, as shown in 1 in Figure 3; if the detection band 5 does not develop color, and If the other two detection bands and the quality control band develop color, it means that the sample is positive, and the AVM content in the sample is between 1.0 μg/L and 2.0 μg/L, as shown in 2 in Figure 3; if the detection band 5 and the detection band 6 does not develop color, and the detection band 7 and the quality control band develop color, indicating that the sample is positive, and the AVM content in the sample is between 2.0 μg/L and 5.0 μg/L, as shown in 3 in Figure 3; if the detection band 5, 6, and 7 do not develop color, and only the quality control band develops color, which means that the sample is positive, and the AVM content in the sample reaches or exceeds 5.0 μg/L, as shown in Figure 3 4; if the detection band does not develop color , and the quality control strip does not develop color, it means that the test strip has expired and cannot be used. It must be replaced with a new test strip and tested again, as shown in 5 in Figure 3.

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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