CN111751535B - Test strip for detecting endosulfan and application thereof - Google Patents
Test strip for detecting endosulfan and application thereof Download PDFInfo
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- CN111751535B CN111751535B CN202010625302.3A CN202010625302A CN111751535B CN 111751535 B CN111751535 B CN 111751535B CN 202010625302 A CN202010625302 A CN 202010625302A CN 111751535 B CN111751535 B CN 111751535B
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- endosulfan
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- RDYMFSUJUZBWLH-SVWSLYAFSA-N endosulfan Chemical compound C([C@@H]12)OS(=O)OC[C@@H]1[C@]1(Cl)C(Cl)=C(Cl)[C@@]2(Cl)C1(Cl)Cl RDYMFSUJUZBWLH-SVWSLYAFSA-N 0.000 title claims abstract description 86
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- 244000299906 Cucumis sativus var. sativus Species 0.000 claims abstract 2
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- JLQUFIHWVLZVTJ-UHFFFAOYSA-N carbosulfan Chemical compound CCCCN(CCCC)SN(C)C(=O)OC1=CC=CC2=C1OC(C)(C)C2 JLQUFIHWVLZVTJ-UHFFFAOYSA-N 0.000 description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a test strip for detecting endosulfan and application thereof. The test strip comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), wherein the reaction membrane is provided with a detection line (5) coated with a endosulfan hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a endosulfan monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting the endosulfan residue in vegetables and fruits such as cucumber, apple and the like by using the endosulfan test strip. The test strip provided by the invention has the characteristics of simplicity in operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a test strip for detecting endosulfan and application thereof, in particular to a colloidal gold test strip for detecting endosulfan, which is particularly suitable for detecting endosulfan residues in vegetables and fruits such as cucumbers, apples and the like.
Background
Sudan is an artificially synthesized organic chlorine compound and is widely used as pesticide. The medicine has the advantages of wide insecticidal range, low price, low toxicity to bees and the like, and is widely applied to various crops. The production of endosulfan begins in the 90 s of the last century in China, 47 endosulfan products are registered for use by 2011, and the endosulfan products relate to 43 families of medicine enterprises and are called as the second large-scale production country of the medicine. The medicine has long effective period, biological enrichment effect, and great harm to fish, shellfish, etc., and accumulated toxicity to mammal. There are a number of documents reporting the detection of endosulfan residue in crops such as vegetables, fruits and the like.
Currently, 70 or more countries worldwide prohibit the production and use of endosulfan. According to 1586 announcements jointly issued by five committees of the department of agriculture and the like in China, the registration of the endosulfan in apple trees and tea trees is cancelled. This means that the endosulfan must be used on the withdrawn crop and the production and use of endosulfan will be phased out.
The research method of the endosulfan at home and abroad mostly adopts solid phase extraction and gas chromatography detection. Compared with the two methods, the immunochemistry detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like for drug detection. The method for preparing the endosulfan artificial antigen is applied to a rapid detection test strip, has short time, simple operation and low cost, and is suitable for detecting samples in a basic unit in batches.
Disclosure of Invention
The invention aims to provide a endosulfan residue detection test strip which has high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting the endosulfan residue comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7); the reaction membrane is provided with a detection line (5) coated with a endosulfan hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a endosulfan monoclonal antibody-colloidal gold marker.
The endosulfan hapten-carrier protein conjugate is obtained by coupling a endosulfan hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine and human serum albumin.
The endosulfan monoclonal antibody is prepared by taking a endosulfan hapten-carrier protein conjugate as an immunogen and is secreted by a endosulfan monoclonal antibody hybridoma cell strain; the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
The sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7), and the conjugate releasing pad 1/3-1/2 is covered below the sample absorbing pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the above test strip, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a endosulfan monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a endosulfan hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
Specifically, the method comprises the following steps:
1) Hapten preparation: the thiodanol and 1, 4-dioxane and other substances undergo a series of chemical reactions to obtain thiodan hapten;
2) Coupling the endosulfan hapten with carrier protein to obtain a endosulfan hapten-carrier protein conjugate;
3) Immunizing a mouse by using a endosulfan hapten-carrier protein conjugate, and obtaining a endosulfan monoclonal hybridoma cell strain by fusing and screening spleen cells and myeloma cells of the mouse;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;
5) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;
6) Adding the endosulfan monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain a endosulfan monoclonal antibody-colloidal gold marker;
7) Spraying the endosulfan monoclonal antibody-colloidal gold marker on a conjugate release pad, baking at 37 ℃ for 1h, taking out, and storing in a dry environment for standby;
8) Coating a thiodan hapten-carrier protein conjugate on a reaction membrane to form a detection line, and coating a goat anti-mouse anti-antibody on the reaction membrane to form a quality control line;
9) Soaking the sample absorption pad in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction) with pH of 7.2 and 0.1mol/L for 2h, and drying at 37deg.C for 2h;
10 The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, the sample absorbing pad covers the conjugate releasing pad, finally, the sample absorbing pad is cut into small strips with the width of 3mm, a plastic box is added, and the sample absorbing pad can be stored for 12 months at the temperature of 4-30 ℃ after vacuum packaging.
The invention also provides a method for detecting the endosulfan residue in vegetables and fruits by using the test strip, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The rapid detection test strip of the endosulfan adopts a highly specific antibody antigen reaction and competitive inhibition immunochromatography analysis technology, a endosulfan monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and endosulfan in a sample is combined with the endosulfan monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a drug-antibody-colloidal gold marker. The medicine in the sample competes with the endosulfan hapten-carrier protein conjugate on the reaction membrane detection line for combining with the endosulfan monoclonal antibody-colloidal gold marker, and whether the endosulfan residue is contained in the sample liquid to be detected is judged according to the existence or the darkness of the red strip of the detection line.
During detection, a sample is dripped into a test strip hole after treatment, when the concentration of the endosulfan in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with the endosulfan hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, and a red strip appears in a detection line (T) and a quality control line (C) respectively; if the concentration of endosulfan in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold-labeled substance will bind to endosulfan entirely, so that no red band will appear at the T-line due to competition reaction with the endosulfan hapten-carrier protein conjugate.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long shelf life. The method for detecting the endosulfan residue by using the test strip is simple, convenient, quick, visual and accurate, has wide application range, low cost and easy popularization and use.
Drawings
FIG. 1 is a diagram showing the synthesis of endosulfan hapten.
Fig. 2 is a schematic diagram of a cross-sectional structure of the test strip.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.
Example 1 preparation of Sudan test strips
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a endosulfan monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a endosulfan hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the PVC bottom plate which are prepared in the steps 1) and 2) into the test strip.
The following is a stepwise detailed description:
1. preparation of endosulfan hapten
Taking 3.6g of endosulfan alcohol, adding 100ml of 1, 4-dioxane for dissolution, fully stirring, adding 3.0g of molecular sieve, adding 1.02g of succinic semialdehyde, fully stirring, introducing hydrochloric acid gas, and stirring at the dark room temperature for 5 hours. Stopping the reaction, adding 0.5mol/L NaOH solution, sufficiently shaking 200ml, adding 200ml ethyl acetate, sufficiently shaking, standing, separating out water phase, washing the organic phase with 80ml water, standing after shaking, separating out water phase, drying and evaporating the organic phase with anhydrous sodium sulfate, loading on a silica gel column, eluting with dichloromethane/methanol (v/v, 10/1), and purifying to obtain 2.1g of succinyland hapten product with a yield of 45.75%.
2. Preparation of immunogens
Taking 16mg of sulfosuccinum acetal, adding 1ml of DMF for dissolution, adding 18.7mg of NHS and 33mg of DDC, and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M PB buffer solution for dissolution to obtain solution B, dripping solution A into solution B, reacting at 4 ℃ for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing the solution three times a day to obtain a thiodan-BSA conjugate which is an immunogen, and preserving at-20 ℃ for later use.
3. Preparation of coating Material
Taking 10mg of Succinum acetal, adding 1ml of DMSO for dissolution, adding 6.7mg of NHS and 16mg of EDC, and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of egg serum albumin (OVA), adding 0.05M PB buffer solution for dissolution to obtain solution B, dripping solution A into solution B, reacting at 4 ℃ for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing the solution three times a day to obtain the endosulfan-OVA conjugate, namely the coating antigen, and preserving at-20 ℃ for later use.
4. Preparation of endosulfan monoclonal antibodies
(1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.
(2) Cell fusion and cloning
Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting an indirect competition ELISA method, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation
The hybridoma cells were prepared into 1X 10 by using a frozen stock solution 6 Cell suspensions/ml were stored in liquid nitrogen for a long period of time. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, so that the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
6. Preparation of endosulfan monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100ml in a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
(2) Preparation of endosulfan monoclonal antibody-colloidal gold marker
Under the magnetic stirring, the pH value of the colloidal gold is regulated to 7.0 by using 0.2mol/L potassium carbonate solution, the endosulfan monoclonal antibody is added into the colloidal gold solution according to the standard of adding 20-50 mug into each milliliter of the colloidal gold solution, the mixture is continuously stirred and mixed for 30min, 10% BSA is added, and the final concentration of the endosulfan monoclonal antibody in the colloidal gold solution is 1% (volume fraction) and is kept stand for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.
Reconstitution buffer: 0.02 to 0.1 percent (mass fraction) of casein, 0.05 to 0.2 percent (mass fraction) of tween-80 and 0.02mol/L of phosphate buffer solution with pH of 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer is 0.5%), having a pH of 7.2 and 0.5mol/L, and was uniformly soaked for 1h and baked at 37℃for 3 h. Uniformly spraying the prepared endosulfan monoclonal antibody-colloidal gold marker on a conjugate release pad by using an isolow film spraying instrument, spraying 0.01ml endosulfan monoclonal antibody-colloidal gold marker on each 1cm conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.
8. Preparation of reaction film
The endosulfan hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the endosulfan hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the endosulfan hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an isolow spot film tester, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/ml with 0.01mol/L phosphate buffer at pH7.4, and coated on a quality control line (C line) on a nitrocellulose membrane in an isolow spot film apparatus in an amount of 1.0. Mu.l/cm. And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
9. Preparation of sample absorbent pad
The sample absorbing pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2 hours, and is baked at 37 ℃ for 2 hours for standby.
10. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3mm by a machine, and placing the test paper strip in a special plastic card, wherein the test paper strip can be stored for 12 months at the temperature of 4-30 ℃.
Example 2 detection of endosulfan residue in samples
1. Detection is carried out by using a test strip
Sucking the sample solution to be detected by a suction pipe, vertically dripping 3 drops of the sample solution into a sample adding hole, starting timing when the liquid flows, reacting for 5-10 min, and judging the result.
2. Analyzing the detection result
The results were read by a colloidal gold analyzer (abbreviated as "analyzer"):
negative (-). Indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
invalidation: indicating that retesting is required.
Example 3 sample detection example
1. Limit of detection test
Taking blank cucumber and apple samples, respectively adding endosulfan to the blank cucumber and apple samples with final concentrations of 50, 100 and 200 mug/kg, taking test strips for detection, and repeating the measurement for three times for each sample.
When the test strip is used for detecting cucumber and apple samples, the analyzer shows negative when the adding concentration of the endosulfan is 50 mug/kg; the analyzer showed positive when the endosulfan addition concentration was 100,200 μg/kg.
2. False positive rate and false negative rate test
Taking 20 parts of cucumber and apple positive samples with the known endosulfan content of 100 mug/kg and 20 parts of cucumber and apple negative samples with the known endosulfan content of less than 50 mug/kg, detecting by using three batches of test strips, and calculating the negative-positive rate. The results are shown in Table 1 and Table 2.
TABLE 1 cucumber test sample results
Table 2 apple test sample results
The results show that: when 3 batches of produced test strips are used for detecting positive cucumber and apple samples, the results are positive, the coincidence rate of the positive samples is 100%, and the false negative rate is 0; when 20 negative cucumber and apple samples are detected, the results are all negative, and the negative coincidence rate is 100% and the false positive rate is 0. The test strip for detecting the endosulfan can be used for rapidly detecting the endosulfan residue in the cucumbers and apples.
3. Specificity test
The thiodan test strip is used for detecting drugs such as carbosulfan, methamidophos, bromomethane and the like with the concentration of 500 mug/kg. The result shows that the quality control line and the detection line of the test strip are both developed and negative. The test strip has no cross reaction to drugs such as carbosulfan, methamidophos, bromomethane and the like with the concentration of 500 mug/kg.
Claims (7)
1. The test strip for detecting the endosulfan comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), and is characterized in that the reaction membrane is provided with a detection line (5) coated with a endosulfan hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with a endosulfan monoclonal antibody-colloidal gold marker, and the endosulfan hapten is prepared by the following steps:
taking 3.6g of endosulfan alcohol, adding 100ml of 1, 4-dioxane for dissolution, fully stirring, adding 3.0g of molecular sieve, adding 1.02g of succinic semialdehyde, fully stirring, introducing hydrochloric acid gas, and stirring at a dark room temperature for 5 hours; stopping the reaction, adding 0.5mol/L NaOH solution, sufficiently shaking 200ml, adding 200ml ethyl acetate, sufficiently shaking, standing, separating out water phase, washing the organic phase with 80ml water, standing after shaking, separating out water phase, drying and evaporating the organic phase with anhydrous sodium sulfate, loading on a silica gel column, eluting and purifying with dichloromethane/methanol with the volume ratio of 10/1 to obtain 2.1g of succinyl thiodan hapten product, wherein the yield is 45.75%;
the molecular structural formula of the endosulfan hapten is as follows:
2. the test strip according to claim 1, wherein the sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7).
3. The test strip of any one of claims 1-2, wherein the conjugate release pad is 1/3 to 1/2 covered under the sample absorbent pad.
4. The test strip of claim 1, wherein the endosulfan hapten-carrier protein conjugate is obtained by coupling a endosulfan hapten with a carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine, or human serum albumin.
5. The test strip of claim 1, wherein said endosulfan monoclonal antibody is prepared by using a endosulfan hapten-carrier protein conjugate as an immunogen, and said goat anti-mouse antibody is prepared by immunizing a goat with a murine antibody.
6. A method of preparing the test strip of any one of claims 1-5, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a endosulfan monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a endosulfan hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
7. A method for detecting the endosulfan residue in vegetables and fruits such as cucumber, apple, etc. comprises the following steps:
1) Sample pretreatment;
2) Detecting with the test strip of any one of claims 1-5;
3) And analyzing the detection result.
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