CN110684111A - Preparation and application of myclobutanil monoclonal antibody - Google Patents
Preparation and application of myclobutanil monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a preparation method of a myclobutanil monoclonal antibody, and the application of the myclobutanil monoclonal antibody in a myclobutanil residue detection test strip and an enzyme linked immunosorbent assay kit. The test strip comprises: the kit 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 myclobutanil 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 myclobutanil monoclonal antibody-colloidal gold marker. The kit comprises: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a myclobutanil standard solution, a myclobutanil antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is myclobutanil coupled antigen, and the enzyme conjugate is an enzyme-labeled myclobutanil antibody. The test strip and the enzyme linked immunosorbent assay kit provided by the invention have the characteristics of simple operation, high sensitivity, high detection speed, low cost and the like, and are suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a preparation method and application of a myclobutanil monoclonal antibody, in particular to a preparation method of the myclobutanil monoclonal antibody, which is particularly suitable for detecting myclobutanil residues in crops such as vegetables, fruits and the like.
Background
Myclobutanil as an ergosterol biosynthesis inhibitor has the characteristics of long lasting period, safety to crops, drug effect number, strong systemic property and the like, and can effectively prevent and treat common diseases in the growth process of a plurality of vegetables and fruits, such as: scab, rust and powdery mildew of pears and apples; powdery mildew of tobacco; net blotch, barley stripe disease, wheat stem blight, net stinking smut, hard smut and loose smut of wheat; powdery mildew and black rot of grapes; rot, stain, brown rust, powdery mildew, etc. of stone fruit. Myclobutanil, which is a pesticide, is used on crops, and is mostly converted, but some pesticide still remains in the crops, and if the pesticide remaining in food is taken for a long time, the health of human bodies is threatened, so that the residual quantity of the pesticide must be considered. Therefore, it is necessary to detect the pesticide.
Most of the existing methods for detecting myclobutanil are instrument methods, have high instrument cost, long detection time and complex operation, and do not meet the high-efficiency and convenient detection requirements of basic laboratories, so the monoclonal antibody of myclobutanil prepared by the invention is applied to the preparation of rapid detection products such as colloidal gold test strips, enzyme linked immunosorbent assay kits, can adapt to the detection requirements of basic laboratories, and is beneficial to improving the supervision means and the supervision level.
Disclosure of Invention
The invention aims to provide a preparation method of a myclobutanil monoclonal antibody, and the application of the myclobutanil monoclonal antibody in a myclobutanil residue detection test strip and an enzyme linked immunosorbent assay kit.
The myclobutanil monoclonal antibody provided by the invention is prepared by taking a myclobutanil hapten-carrier protein conjugate as an immunogen, and is obtained by secreting a myclobutanil monoclonal antibody hybridoma cell strain; the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody; the myclobutanil hapten-carrier protein conjugate is obtained by coupling myclobutanil hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein and human serum albumin.
The test strip for detecting myclobutanil 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 myclobutanil 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 myclobutanil monoclonal antibody-colloidal gold marker.
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.
The enzyme linked immunosorbent assay kit for detecting myclobutanil residue provided by the invention comprises: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a myclobutanil standard solution, a myclobutanil antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is myclobutanil coupled antigen, and the enzyme conjugate is an enzyme-labeled myclobutanil antibody.
The specific antibody of myclobutanil is prepared by taking myclobutanil coupled antigen as immunogen, and the specific antibody of myclobutanil can be monoclonal antibody of myclobutanil or polyclonal antibody of myclobutanil, wherein the monoclonal antibody of myclobutanil is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and myclobutanil antibody.
The rapid detection test strip for myclobutanil adopts the high-specificity antibody-antigen reaction and competitive inhibition immunochromatography analysis technology, the myclobutanil monoclonal antibody-colloidal gold marker is fixed on the conjugate release pad, and myclobutanil in a sample is combined with the myclobutanil monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the drug-antibody-colloidal gold marker. The drug in the sample and the myclobutanil hapten-carrier protein conjugate on the reaction membrane detection line compete to combine with the myclobutanil monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains myclobutanil residue is judged according to the fact that the red strip of the detection line has none or light color.
The kit adopts a direct competition ELISA method, pre-coats coupling antigen on an enzyme label plate microporous strip, the residual myclobutanil in a sample and the coupling antigen pre-coated on the enzyme label plate microporous strip compete for an enzyme conjugate resisting myclobutanil, the TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of residual myclobutanil contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple to obtain the residual myclobutanil in the sample.
The test strip and the kit have 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 residual myclobutanil by using the test strip and the kit 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 myclobutanil hapten.
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 Myclobutanil monoclonal antibody
1. Preparation of myclobutanil hapten
Taking 1.79g of 2- (4-chlorphenyl) -2-cyanoacetaldehyde, adding 20ml of dimethyl sulfoxide for dissolving, adding 3.42g of dibromomethane, fully stirring, adding 0.4g of phase transfer catalyst and 0.8g of sodium hydroxide, fully stirring for 20min, reacting at constant temperature of 60 ℃ for 12h, stopping the reaction, cooling to room temperature, adding 6mol/L hydrochloric acid for adjusting the pH value to 7, adding 100ml of dichloromethane and 80ml of water, shaking, standing, separating a water phase, concentrating and evaporating an organic phase, and recrystallizing with 80ml of n-hexane/ethyl acetate (v/v, 10/1) to obtain 1.3g of an intermediate compound b, wherein the yield is 47.79%;
taking 1.3g of the compound b, adding 60ml of DMF (dimethyl formamide) for dissolution, adding 0.64g of potassium hydroxide and 0.21g of anhydrous sodium iodide, fully stirring, adding 0.41g of triazole, heating at 80 ℃, stirring for 5h, stopping the reaction, adding 150ml of water and 120ml of ethyl acetate, extracting, washing with water, concentrating and evaporating to dryness, loading on a silica gel column, and eluting and separating dichloromethane/methanol (v/v, 5/1) to obtain 0.8g of myclobutanil hapten with the yield of 64.33%.
2. Preparation of immunogens
Dissolving 9.7mg of aldehydic myclobutanil in 1ml of ethanol to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M CB buffer solution for dissolving to obtain B solution, dripping A solution into the B solution, reacting for 12h at 4 ℃, adding 5mg of sodium borohydride, continuously stirring for 2h, dialyzing and purifying for 3 days by 0.02M PBS, changing the solution three times every day to obtain myclobutanil-BSA conjugate which is immunogen, and storing at-20 ℃ for later use.
3. Preparation of coating antigen
Dissolving 5.8mg of aldehyde group myclobutanil in 1ml of ethanol to obtain a hapten solution A; dissolving egg serum albumin (OVA)50mg in 0.05M CB buffer solution to obtain solution B, dripping A solution into the solution B, reacting at 4 ℃ for 12h, adding sodium borohydride 3.7mg, continuously stirring for 2h, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution three times every day to obtain myclobutanil-BSA conjugate which is coating antigen, and storing at-20 ℃ for later use.
4. Preparation of myclobutanil 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
Making 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.
Example 2 preparation of Myclobutanil residue test strip
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with a myclobutanil monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a myclobutanil 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.
1. Preparation of myclobutanil 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 myclobutanil 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 myclobutanil monoclonal antibody into the colloidal gold solution according to the standard of adding 20-50 mu g of myclobutanil monoclonal antibody into each milliliter of colloidal gold solution, continuously stirring and uniformly mixing for 30min, adding 10% BSA (bovine serum albumin) to enable the final concentration of the BSA in the colloidal gold solution to be 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.
2. 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. The prepared myclobutanil monoclonal antibody-colloidal gold marker is evenly sprayed on a conjugate release pad by an Isoflow film spraying instrument, 0.01ml of myclobutanil monoclonal antibody-colloidal gold marker is sprayed on each 1cm of conjugate release pad, and then the myclobutanil monoclonal antibody-colloidal gold marker is placed in an environment with the temperature of 37 ℃ (the humidity is less than 20%) for 60min and then taken out, and is placed in a dry environment (the humidity is less than 20%) for storage and standby.
3. Preparation of the reaction film
Coating the myclobutanil 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 myclobutanil hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the myclobutanil 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.
4. 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.
5. 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 3 preparation of Myclobutanil residue ELISA kit
1. Preparation of enzyme conjugates
Taking a goat as an immune animal and taking a myclobutanil monoclonal antibody as an immunogen to immunize a goat without a pathogen to obtain the myclobutanil antibody. Coupling the myclobutanil antibody with horseradish peroxidase (HRP) to obtain an enzyme conjugate.
2. Preparation of ELISA plates
Diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 200 mu l of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
3. The enzyme linked immunosorbent kit comprises the following components:
(1) an ELISA plate coated with myclobutanil coupling antigen;
(2) 6 bottles of myclobutanil standard solution;
(3) a myclobutanil antibody labeled with horseradish peroxidase;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
Claims (4)
1. The myclobutanil monoclonal antibody is characterized in that the monoclonal antibody is prepared by taking a myclobutanil hapten-carrier protein conjugate as an immunogen, and the preparation method of the hapten comprises the following steps:
taking 1.79g of 2- (4-chlorphenyl) -2-cyanoacetaldehyde, adding 20ml of dimethyl sulfoxide for dissolving, adding 3.42g of dibromomethane, fully stirring, adding 0.4g of phase transfer catalyst and 0.8g of sodium hydroxide, fully stirring for 20min, reacting at constant temperature of 60 ℃ for 12h, stopping the reaction, cooling to room temperature, adding 6mol/L hydrochloric acid for adjusting the pH value to 7, adding 100ml of dichloromethane and 80ml of water, shaking, standing, separating a water phase, concentrating and evaporating an organic phase, and recrystallizing with 80ml of n-hexane/ethyl acetate (v/v, 10/1) to obtain 1.3g of an intermediate compound b, wherein the yield is 47.79%;
taking 1.3g of the compound b, adding 60ml of DMF (dimethyl formamide) for dissolution, adding 0.64g of potassium hydroxide and 0.21g of anhydrous sodium iodide, fully stirring, adding 0.41g of triazole, heating at 80 ℃, stirring for 5h, stopping the reaction, adding 150ml of water and 120ml of ethyl acetate, extracting, washing with water, concentrating and evaporating to dryness, loading on a silica gel column, and eluting and separating dichloromethane/methanol (v/v, 5/1) to obtain 0.8g of myclobutanil hapten with the yield of 64.33%.
2. The myclobutanil monoclonal antibody of claim 1, wherein the molecular structural formula of the myclobutanil hapten is:
3. the myclobutanil monoclonal antibody of claim 1, wherein the monoclonal antibody is used for preparing a test strip for detecting myclobutanil, the test strip comprises: the kit 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 myclobutanil 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 myclobutanil monoclonal antibody-colloidal gold marker.
4. The myclobutanil monoclonal antibody of claim 1, wherein the monoclonal antibody is used for preparing an enzyme linked immunosorbent assay kit for detecting myclobutanil, the kit comprises: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a myclobutanil standard solution, a myclobutanil antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is myclobutanil coupled antigen, and the enzyme conjugate is an enzyme-labeled myclobutanil antibody.
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| CN113702628A (en) * | 2020-05-20 | 2021-11-26 | 四川精卫食品检测科技有限公司 | Myclobutanil colloidal gold detection kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10248565A (en) * | 1997-03-18 | 1998-09-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of myclobutanil compound, antibody and measurement |
| CN109298191A (en) * | 2018-09-30 | 2019-02-01 | 云南省烟草农业科学研究院 | A kind of nitrile bacterium azoles colloidal gold colloidal gold detection test paper strip and the preparation method and application thereof |
-
2019
- 2019-10-15 CN CN201910976783.XA patent/CN110684111A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10248565A (en) * | 1997-03-18 | 1998-09-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of myclobutanil compound, antibody and measurement |
| CN109298191A (en) * | 2018-09-30 | 2019-02-01 | 云南省烟草农业科学研究院 | A kind of nitrile bacterium azoles colloidal gold colloidal gold detection test paper strip and the preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| ANDRÁS SZÉKÁCS & BRUCE D. HAMMOCK: "Development of an Enzyme-Linked Immunosorbent Assay for the Detection of the Triazole Fungicide Myclobutanil", 《J. AGRIC. FOOD CHEM》 * |
| FERENC SZURDOKI ET AL.: "Synthesis of haptens and protein conjugates for the development of immunoassays for the insect growth regulator fenoxycarb", 《J AGRIC FOOD CHEM》 * |
| JUAN J MANCLÚS ET AL.: "Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices", 《J AGRIC FOOD CHEM》 * |
| 唐除痴等: "《农药化学》", 31 March 1998, 南开大学出版社 * |
| 张玉芬，王华主编: "《农药残留检测与安全性评价》", 31 March 2013, 黑龙江大学出版社 * |
| 沙家骏等主编: "《国外新农药品种手册》", 31 October 1992, 化学工业出版社 * |
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| CN113702628A (en) * | 2020-05-20 | 2021-11-26 | 四川精卫食品检测科技有限公司 | Myclobutanil colloidal gold detection kit and application thereof |
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