CN111751535A - Test strip for detecting endosulfan and application thereof - Google Patents
Test strip for detecting endosulfan and application thereof Download PDFInfo
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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 endosulfan residues in vegetables and fruits such as cucumbers, apples and the like by applying the endosulfan test strip. The test strip provided by the invention has the characteristics of simple 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
Endosulfan is an artificially synthesized organochlorine compound and is widely used as a 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. China began to produce endosulfan in the last 90 th century, and by 2011, 47 endosulfan products which are registered and used relate to 43 families of medicine enterprises, which are called the second major producing country of the medicine. The medicine has long shelf life, biological enrichment effect, great harm to fish and shellfish, and cumulative toxicity to mammal. There have been a number of reports in the literature on the detection of endosulfan residues in crops such as vegetables and fruits.
Currently, there are 70 countries worldwide that prohibit the production and use of endosulfan. According to the notice No. 1586 issued by five committees of Ministry of agriculture in China, the registration of endosulfan on apple trees and tea trees is cancelled. This means that endosulfan must not continue to be used on the withdrawn crop and will phase out the production and use of endosulfan.
Solid phase extraction and gas chromatography detection are mostly adopted in the research methods of endosulfan at home and abroad. Compared with the two methods, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like for the detection of the medicine. The invention discloses a method for preparing an artificial antigen of endosulfan, which is applied to a rapid detection test strip, has the advantages of short time, simple operation and low cost, and is suitable for detecting samples in bulk by a basic unit.
Disclosure of Invention
The invention aims to provide the endosulfan residue detection test strip which is high in sensitivity, simple to operate, low in cost and short in detection time.
The test strip for detecting endosulfan residue provided by the invention 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 thiodan 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 thiodan monoclonal antibody-colloidal gold marker.
The endosulfan hapten-carrier protein conjugate is obtained by coupling endosulfan hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
The endosulfan monoclonal antibody is prepared by taking endosulfan hapten-carrier protein conjugate as immunogen and is obtained by secreting endosulfan monoclonal antibody hybridoma cell strain; the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
The sample absorption pad (1), the conjugate release pad (2), the reaction film (3) and the water absorption pad (4) are sequentially adhered to the bottom plate (7), and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
The bottom plate can be a PVC bottom plate or other hard non-absorbent 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 absorption pad is absorbent paper; the reaction membrane can be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the test strip, which comprises 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) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
Specifically, the steps include:
1) hapten preparation: carrying out a series of chemical reactions on the thiodan alcohol, 1, 4-dioxane and other substances to obtain a thiodan hapten;
2) coupling the endosulfan hapten with carrier protein to obtain an endosulfan hapten-carrier protein conjugate;
3) immunizing a mouse by using the endosulfan hapten-carrier protein conjugate, and fusing and screening splenocytes and myeloma cells of the mouse to obtain a endosulfan monoclonal hybridoma cell strain;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
6) adding the endosulfan monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain an endosulfan monoclonal antibody-colloidal gold marker;
7) spraying the endosulfan monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for later use;
8) coating the endosulfan hapten-carrier protein conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line;
9) soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), pH7.2, 0.1mol/L phosphate buffer solution for 2h, and drying at 37 deg.C for 2 h;
10) and sequentially adhering a sample absorption pad, a conjugate release pad, a reaction film and a water absorption pad on the bottom plate, covering the conjugate release pad with the sample absorption pad, finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.
The invention also aims to provide a method for detecting endosulfan residues in vegetables and fruits by using the test strip, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using a test strip;
(3) and analyzing the detection result.
The rapid test strip for the endosulfan adopts a highly specific antibody-antigen reaction and competitive inhibition immunochromatography analysis technology, the endosulfan monoclonal antibody-colloidal gold marker is fixed on the 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 drug in the sample and the thiodan hapten-carrier protein conjugate on the reaction membrane detection line compete to combine with the thiodan monoclonal antibody-colloidal gold marker, and whether the liquid sample to be detected contains thiodan residues or not is judged according to the existence of red strips or the color depth of the red strips on the detection line.
During detection, a sample is treated and then dropped into a test strip hole, when the concentration of endosulfan in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with 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); if the concentration of endosulfan in the sample is equal to or above the detection limit, the monoclonal antibody-colloidal gold label will bind to all endosulfan, and thus no red band will appear at the T-line due to the competitive reaction without binding to 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 quality guarantee period. The method for detecting the endosulfan residue by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a diagram of the synthesis of endosulfan hapten.
FIG. 2 is a schematic cross-sectional view of a test strip.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of the test strip for endosulfan detection
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) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a PVC base plate to form the test strip.
The following steps are detailed:
1. preparation of endosulfan hapten
Taking 3.6g of sudan alcohol, adding 100ml of 1, 4-dioxane for dissolving, fully stirring, then adding 3.0g of molecular sieve, adding 1.02g of succinic semialdehyde, fully stirring, introducing hydrochloric acid gas, and stirring for 5 hours at room temperature in a dark place. Stopping the reaction, adding 0.5mol/L NaOH solution, fully shaking 200ml, adding 200ml of ethyl acetate, fully shaking, standing, separating a water phase, washing an organic phase with 80ml of water, standing after shaking, separating the water phase, drying and evaporating the organic phase with anhydrous sodium sulfate, applying to a silica gel column, eluting and purifying with dichloromethane/methanol (v/v, 10/1) to obtain 2.1g of the succinyl acetal thiodan hapten product with the yield of 45.75%.
2. Preparation of immunogens
Taking 16mg of succinyl acetal endosulfan, adding 1ml of DMF for dissolving, adding 18.7mg of NHS and 33mg of DDC, and reacting for 3 hours at room temperature to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M PB buffer solution for dissolving to obtain solution B, dripping A solution into the solution B, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02M PBS, and changing the solution three times every day to obtain a endosulfan-BSA conjugate which is an immunogen and is stored at-20 ℃ for later use.
3. Preparation of coating antigen
Dissolving Succinal acetal endosulfan 10mg in DMSO 1ml, adding NHS6.7mg and EDC16mg, and reacting at room temperature for 3 hr to obtain hapten solution A; dissolving egg serum albumin (OVA)50mg in 0.05M PB buffer solution to obtain solution B, dripping A solution into solution B, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing solution three times per day to obtain endosulfan-OVA conjugate, which is coating antigen, and storing at-20 deg.C.
4. Preparation of endosulfan monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting an indirect competitive ELISA method, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Preparing hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per ml, preserved for long periods in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
6. Preparation of endosulfan monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, placing 100ml into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous stirring at high temperature, stirring at constant speed, heating until the solution is bright red, cooling to room temperature, recovering to original volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating material.
(2) Preparation of endosulfan monoclonal antibody-colloidal gold marker
Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.0 by using 0.2mol/L potassium carbonate solution, adding the endosulfan monoclonal antibody into the colloidal gold solution according to the standard of adding 20-50 mu g of endosulfan monoclonal antibody into each milliliter of the colloidal gold solution, continuously stirring and uniformly mixing for 30min, adding 10% BSA to ensure that the final concentration of the BSA in the colloidal gold solution is 1% (volume fraction), and standing for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.
Redissolving buffer solution: 0.02mol/L phosphate buffer solution containing casein 0.02-0.1% (mass fraction), tween-800.05-0.2% (mass fraction) and pH7.2.
7. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5mol/L phosphate buffer containing bovine serum albumin (concentration of bovine serum albumin in buffer is 0.5%), pH7.2, and the solution was soaked for 1h, and then baked at 37 ℃ for 3 h. And uniformly spraying the prepared endosulfan monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01ml endosulfan monoclonal antibody-colloidal gold marker on every 1cm of conjugate release pad, placing in an environment at 37 ℃ (humidity is less than 20%) for 60min, taking out, and placing in a dry environment (humidity is less than 20%) for storage.
8. Preparation of the reaction film
Coating the endosulfan hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.
Coating process: 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 Isoflow point membrane instrument, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/ml with 0.01mol/L, pH7.4 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 1.0. mu.l/cm using an Isoflow dot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
9. Preparation of sample absorbent pad
The sample absorption pad is soaked in 0.5% bovine serum albumin (volume fraction), pH7.2, 0.1mol/L phosphate buffer solution for 2h, and then baked at 37 ℃ for 2h 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 binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC soleplate, and the tail end of the water absorption pad is aligned with the tail end of the PVC soleplate; the reaction membrane 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 vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; the test paper strip is cut into small strips with the width of 3mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃.
EXAMPLE 2 detection of endosulfan residue in samples
1. Detection with test strip
And (3) sucking the sample solution to be detected by using a suction pipe, vertically and dropwise adding the sample solution to be detected into the sample adding hole, starting timing when the liquid flows, reacting for 5-10 min, and judging the result.
2. Analyzing the results of the detection
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;
and (4) invalidation: indicating that retesting is required.
Example 3 sample testing example
1. Limit of detection test
Taking blank cucumber and apple samples, respectively adding endosulfan into the samples until the final concentrations are 50, 100 and 200 mu g/kg, taking test strips for detection, and repeatedly measuring each sample for three times.
When the test paper is used for detecting cucumber and apple samples, when the added concentration of endosulfan is 50 mug/kg, the analyzer shows negative; when the added concentration of endosulfan is 100 and 200 mug/kg, the analyzer shows positive.
2. Test for false positive and false negative rates
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 content of less than 50 mug/kg are respectively taken, three batches of test strips are used for detection, and the negative and positive rates are calculated. The results are shown in tables 1 and 2.
TABLE 1 cucumber test sample results
TABLE 2 apple test sample results
The results show that: when 3 test strips produced in batches are used for detecting positive cucumber and apple samples, the results are all positive, and the coincidence rate of the positive samples is 100 percent, 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 percent and the false positive rate is 0. The test strip for detecting endosulfan can be used for rapidly detecting endosulfan residues in cucumbers and apples.
3. Specificity test
The Sudan test paper is used for detecting 500 mu g/kg carbosulfan, methamidophos, methyl bromide and other medicaments. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to 500 mu g/kg carbosulfan, methamidophos, methyl bromide and other medicaments.
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 endosulfan hapten-carrier protein conjugate and a quality control line (6) coated with goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with endosulfan monoclonal antibody-colloidal gold marker, and the preparation method of the endosulfan hapten is as follows:
taking 3.6g of sudan alcohol, adding 100ml of 1, 4-dioxane for dissolving, fully stirring, then adding 3.0g of molecular sieve, adding 1.02g of succinic semialdehyde, fully stirring, introducing hydrochloric acid gas, and stirring for 5 hours at room temperature in a dark place. Stopping the reaction, adding 0.5mol/L NaOH solution, fully shaking 200ml, adding 200ml of ethyl acetate, fully shaking, standing, separating a water phase, washing an organic phase with 80ml of water, standing after shaking, separating the water phase, drying and evaporating the organic phase with anhydrous sodium sulfate, applying to a silica gel column, eluting and purifying with dichloromethane/methanol (v/v, 10/1) to obtain 2.1g of the succinyl acetal thiodan hapten product with the yield of 45.75%.
2. The test strip of claim 1, wherein the sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the absorbent pad (4) are sequentially adhered to the bottom plate (7).
3. The strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.
4. The test strip of claim 1, wherein the endosulfan hapten-carrier protein conjugate is obtained by conjugating endosulfan hapten with carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein, or human serum albumin.
5. The test strip of claim 1, wherein the endosulfan monoclonal antibody is prepared from an endosulfan hapten-carrier protein conjugate as an immunogen, and the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.
6. A method of making 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) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
7. A method for detecting endosulfan residues in vegetables and fruits such as cucumbers, apples and the like comprises the following steps:
1) sample pretreatment;
2) performing a test using the test strip of any one of claims 1-5;
3) and analyzing the detection result.
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| CN112698026A (en) * | 2020-11-18 | 2021-04-23 | 山东勤邦生物技术有限公司 | Test strip for detecting clothianidin and application thereof |
Citations (6)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112698026A (en) * | 2020-11-18 | 2021-04-23 | 山东勤邦生物技术有限公司 | Test strip for detecting clothianidin and application thereof |
| CN112698026B (en) * | 2020-11-18 | 2023-12-12 | 山东勤邦生物技术有限公司 | Test strip for detecting clothianidin and application thereof |
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| CN111751535B (en) | 2023-07-11 |
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