CN112409348A - Preparation method and application of hapten and complete antigen for thiamethoxam - Google Patents
Preparation method and application of hapten and complete antigen for thiamethoxam Download PDFInfo
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Abstract
The invention discloses a thiamethoxam hapten, a complete antigen, a preparation method and application. The structural formula of the thiamethoxam hapten is shown as a formula I. And coupling the thiamethoxam hapten with carrier protein to prepare the thiamethoxam complete antigen. The preparation method is simple and feasible, and the prepared thiamethoxam hapten and thiamethoxam complete antigen can effectively stimulate immunized animals to generate antibodies with high sensitivity and strong specificity, can be used in immunoassay, and can meet the requirement of domestic detection of thiamethoxam residues.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a hapten and a complete antigen for thiamethoxam.
Background
Thiamethoxam belongs to a nicotine pesticide, is an action body of a nicotinic acetylcholine receptor, mainly acts on a postganglionic membrane of an insect nerve, and is combined with the nicotinic acetylcholine receptor to interfere normal conduction of the insect nervous system, so that the blockage of a nerve channel is caused, and a large amount of acetylcholine is accumulated, so that the insect is excited abnormally, and is killed by general spasm and paralysis. Thiamethoxam belongs to a low-toxicity pesticide and has strong contact poisoning, stomach poisoning and systemic effects. The insecticidal composition is mainly used for fruits, vegetables, tea and grain crops, and can effectively prevent and control various pests such as aphids, leafhoppers, plant hoppers, whiteflies, potato beetles, scarab larvae, nematodes, ground beetles, leaf miners and the like, and pests with resistance to various chemical pesticides. Although the thiamethoxam has obvious effect and can effectively control insect pests after being used, the pesticide residue cannot be excluded to threaten human beings and livestock, and related researches show that the nicotine pesticide can interfere the homing of bees and can influence the development of the nervous system of mammals. In recent years, the european union has limited the use of nicotinic insecticides on some crops. The maximum residual quantity limit of thiamethoxam in vegetable common head cabbage is 0.2mg/kg and cucumber is 0.5mg/kg according to the national food safety regulation standard GB2763-2016 (maximum limit of pesticide residue in food); the watermelon content in the fruit is 0.2 mg/kg; the grain contains brown rice 0.1mg/kg and wheat 0.1 mg/kg.
The method for detecting thiamethoxam pesticide residues commonly used at present is a chromatography method, and comprises instrument methods such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS), Supercritical Fluid Chromatography (SFC), Capillary Electrophoresis (CE) and the like, but the methods need expensive instruments and professional operators, and the pretreatment of samples is complex, the cost is high, the time is long, and the rapid detection of a large number of samples cannot be realized, so that the establishment of a rapid, sensitive and cheap thiamethoxam detection method has important significance. Enzyme-linked immunosorbent assay (ELISA) is an extremely high-efficiency, sensitive and rapid detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for field rapid detection of a large number of samples. Establishing an efficient immunological detection method, wherein the preparation of hapten and complete antigen is the first condition. The excellent hapten and complete antigen can excite the immunized animal to generate an antibody with high sensitivity and good specificity.
Disclosure of Invention
The invention aims to provide a thiamethoxam hapten and a thiamethoxam complete antigen which can effectively stimulate immunized animals to generate thiamethoxam antibodies with high sensitivity and strong specificity.
The invention also aims to provide a preparation method of the thiamethoxam hapten and the thiamethoxam complete antigen, which is simple and easy to implement.
The thiamethoxam hapten and the thiamethoxam complete antigen prepared by the method can be established in an immunoassay method of thiamethoxam and applied to residue detection of thiamethoxam in food.
In order to achieve the purpose, the invention adopts the following technical scheme:
the structural formula of the thiamethoxam hapten is shown as a formula I:
the structural formula of the thiamethoxam complete antigen provided by the invention is shown as a formula II:
wherein, Pr in the formula II represents carrier protein, and can be Bovine Serum Albumin (BSA).
The invention also provides a preparation method of the thiamethoxam hapten.
The preparation method of the thiamethoxam hapten comprises the following steps:
under the alkaline condition, the thiamethoxam reacts with 4-mercaptobenzoic acid to obtain the thiamethoxam hapten.
The reaction is carried out in a solvent, which may be N, N-dimethylformamide.
The alkaline condition can be provided by sodium hydroxide, and the molar ratio of the thiamethoxam to the sodium hydroxide is 1:2-3, and can be 1: 3.
The molar ratio of the thiamethoxam to the 4-mercaptobenzoic acid is 1:1.1-1:1.5, and specifically can be 1:1.1
The reaction is carried out under the condition of heating reflux, the temperature of the reaction can be 110 ℃, and the time can be 3-4 hours.
The method further comprises the steps of: and after the reaction is finished, cooling the reaction system to room temperature, adding water to quench the reaction system, adjusting the pH value of the reaction system to be neutral, adding ethyl acetate to stratify, collecting an organic layer, drying and concentrating to obtain a crude thiamethoxam hapten product. Specifically, concentrated hydrochloric acid can be used to adjust the pH value of the reaction system.
The method further comprises the step of purifying the obtained thiamethoxam hapten crude product, and the method comprises the following specific steps: dissolving the crude thiamethoxam hapten product with ethyl acetate again, adding 100-200 silica gel, uniformly stirring, concentrating, removing the solvent, and then carrying out column chromatography separation to obtain a purified thiamethoxam hapten; wherein the eluent for the column chromatographic separation is a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 3: 1.
The invention also provides a preparation method of the thiamethoxam complete antigen.
The thiamethoxam complete antigen is obtained by coupling the thiamethoxam hapten and carrier protein. The thiamethoxam complete antigen can be used as an immunogen or a coating antigen. Wherein, the thiamethoxam complete antigen is used as immunogen, and the carrier protein adopted by the thiamethoxam complete antigen can be Bovine Serum Albumin (BSA); the thiamethoxam complete antigen is used as an envelope antigen, and the carrier protein adopted by the thiamethoxam complete antigen can be egg white albumin (OVA).
The coupling molar ratio of the thiamethoxam hapten to the carrier protein is 20:1-40:1, and specifically can be 20: 1;
the thiamethoxam complete antigen provided by the invention is prepared by coupling carrier protein to carboxyl carbon of thiamethoxam hapten by adopting an activated ester method.
The preparation method comprises the following steps:
a) dissolving thiamethoxam hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature to obtain solution A;
b) dissolving carrier protein in carbonate buffer solution to obtain solution B;
c) mixing the solution A and the solution B and stirring to obtain solution C;
d) and (3) putting the solution C into a dialysis bag, dialyzing the solution C in a phosphate buffer solution, and collecting the solution in the dialysis bag, namely the thiamethoxam complete antigen.
In step a) of the method, the dosage ratio of the thiamethoxam hapten to the dimethylformamide to the dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 20 μmol: 1-2 mL: 60-120 μmol, and specifically can be: 20 mu mol: 1 mL: 60 mu mol; the stirring time may be 18-24 hours,
in step b), the carrier protein may be Bovine Serum Albumin (BSA) or Ovalbumin (OVA).
The dosage ratio of the carrier protein to the carbonate buffer solution is 0.4 mu mol: 3-6mL, and specifically comprises the following components: 0.4. mu. mol: 3 mL.
In step c), the stirring conditions are as follows: stirring at 4-25 deg.C (specifically 4 deg.C) for 10-12 hr (specifically 10 hr).
In step d) of the above method, the dialysis is performed for 6 times by changing the solution every 3 hours.
The invention also provides a thiamethoxam specific antibody prepared from the thiamethoxam complete antigen.
The specific antibody comprises a polyclonal antibody and a monoclonal antibody.
The polyclonal antibody can be obtained by immunizing experimental animals (such as Balb/c mice) with thiamethoxam complete antigen, collecting serum and purifying.
The invention also provides an enzyme-linked immunosorbent assay reagent or a kit for detecting thiamethoxam.
The enzyme-linked immunosorbent assay or kit for detecting thiamethoxam comprises the thiamethoxam complete antigen and the thiamethoxam specific antibody.
The invention also provides application of the thiamethoxam hapten or the thiamethoxam complete antigen.
The application is the application in the preparation of the anti-thiamethoxam specific antibody.
The invention also provides application of the anti-thiamethoxam specific antibody.
The application is selected from at least one of the following aspects:
(1) the application in the detection of thiamethoxam;
(2) the application in preparing the kit for ELISA detection of thiamethoxam;
(3) the application in preparing the colloidal gold test strip of thiamethoxam.
The preparation method is simple and feasible, and the prepared thiamethoxam hapten and thiamethoxam complete antigen can effectively stimulate immunized animals to generate thiamethoxam antibodies with high sensitivity and strong specificity, can be used in immunoassay, and can meet the requirement of domestic residue detection on thiamethoxam.
Drawings
FIG. 1 is a standard inhibition curve of indirect competitive enzyme-linked immunosorbent assay (ICELISA) for detecting thiamethoxam.
Detailed Description
The present invention is described below with reference to specific embodiments, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1 preparation of thiamethoxam hapten of formula I
The following "parts" are all referred to as "mole parts".
1) Dissolving 1 part of thiamethoxam in 3-4 parts of N, N-dimethylformamide, adding 2-3 parts of sodium hydroxide and 1.1 part of 4-mercaptobenzoic acid, heating and refluxing the reaction system at 110 ℃ for 3-4 hours, adding 10 parts of water to quench the reaction when the reaction system is cooled to room temperature, adjusting the pH of the reaction system to be neutral by using concentrated hydrochloric acid, and adding 20 parts of ethyl acetate to stratify. Collecting the organic layer, drying and concentrating to obtain a crude product of the target product.
2) Dissolving 1 part of the target product crude product by using 1 part of ethyl acetate again, adding 1 part of 100-200 silica gel, uniformly stirring, concentrating to remove the solvent, and then carrying out column chromatography separation, wherein an eluent is petroleum ether: ethyl acetate in a volume ratio of 3:1, and concentrating the eluate to give the compound: (E) -4- ((5- ((5-methyl-4- (nitroimino) -1,3, 5-oxadiazin-3-yl) methyl) thiazol-2-yl) thio) benzoic acid, thiamethoxam hapten.
And performing structure identification by nuclear magnetic hydrogen spectrum and high-resolution mass spectrum to obtain the compound which is the target product.
1H NMR(300MHz,CDCl3)δ12.45(s,1H),8.00(d,J＝8.3Hz,2H),7.88(d,J＝8.3Hz,2H),7.66(s,1H),5.01(d,J＝17.5Hz,4H),4.75(s,2H),2.85(s,3H).HRMS calcd for C15H15N5O5S2:[M+H+]410.0587,found 410.0561.
Example 2 preparation of thiamethoxam antigen and thiamethoxam complete antigen of formula II
The thiamethoxam complete antigen was prepared from the thiamethoxam hapten of example 1, comprising the following steps:
1) dissolving thiamethoxam hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature for 18-24 hours to obtain solution A; wherein the dosage ratio of the thiamethoxam hapten to the dimethylformamide to the dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 20 mu mol to 1mL to 60 mu mol in turn;
2) dissolving carrier protein (BSA) in carbonate buffer solution (pH 7.0) to obtain solution B; wherein the dosage ratio of the carrier protein to the carbonate buffer solution is 0.4 mu mol: 3 mL;
3) dripping the A solution into the B solution and stirring for 10-12 hours at 4 ℃ to obtain a C solution;
4) and (3) putting the solution C into a dialysis bag, dialyzing in a phosphate buffer solution, changing the solution once every 3 hours, dialyzing for 6 times totally, and collecting the solution in the dialysis bag, namely the immunogen solution, namely the thiamethoxam complete antigen, and storing at-20 ℃.
Synthesis of thiamethoxam coating antigen:
OVA is used for replacing BSA, and other steps are the same as the above steps to obtain the thiamethoxam coated antigen.
Example 3 identification of the complete antigen of thiamethoxam
Balb/c mice were immunized with the thiamethoxam complete antigen prepared in example 2 according to the protocol in Table 1, and orbital venous serum was collected 7 days after four immunizations and examined to determine whether the prepared thiamethoxam antigen had immunological activity.
TABLE 1 immunization protocol
1) The thiamethoxam-coated antigen (1mg/mL) prepared in example 2 was diluted as shown in Table 2 (the diluent was a sample diluent, the sample diluent was 500mL of PBS (pH 7.5) to which 0.1mL of Tween-20 and 0.5g of gelatin were added), and then the mixture was added to an ELISA plate (100. mu.L per well), incubated at 37 ℃ for 3 hours, removed, and washed with PBST;
2) adding 50 mu L of sample diluent into each hole of the coated plate according to the comparison hole in the table 2, and adding 50 mu L of thiamethoxam standard sample of 200ng/mL into each hole of the inhibition hole;
3) collecting mouse serum, diluting with sample diluent according to the times of Table 2, adding 50 μ L per well, incubating the ELISA plate at 37 deg.C for 0.5h, taking out, and washing with PBST;
4) to the enzyme-labeled wells, 1. mu.g/mL of goat anti-mouse antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd., catalog No.: 111-035-003), 100 muL of each well, then placing the ELISA plate at 37 ℃ for incubation for 0.5h, taking out, washing with PBST, adding 100 muL of developing solution into the ELISA plate, developing at room temperature, stopping the reaction by 50% concentrated sulfuric acid in each well when the concentration difference between the control well and the inhibition well is about 1.0, and reading the light absorption value at 492 nm;
5) the experimental result shows that the light absorption value shows a gradual reduction rule along with the increase of the dilution times of the antigen and the serum. The mouse serum titer was defined as the maximum dilution of antiserum at an OD value of 1.0, and the inhibition ratio ═ [ (control well OD value-inhibition well OD value)/control well OD value ] × 100%, as can be seen from table 2, the mouse serum titer was 4000, when the coated antigen (1.0mg/mL) was diluted at a dilution of 1: 16000 and when the serum is 1:4000, the thiamethoxam has the highest inhibition efficiency of 52.04%.
TABLE 2 mouse serum titer and its inhibition reaction detection on thiamethoxam
Note that C represents a control well in the microplate, I represents a suppression well in the microplate (200ng/mL)
The antigen is used to immunize Balb/c mice, and the monoclonal antibody obtained by screening establishes an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method, the detection range4-101 ng/mL, and the detection limit is 1.95 ng/mL; IC of the method5015ng/mL, and the correlation coefficient of the curve is 0.9993.
TABLE 3 IC of monoclonal antibodies against different nicotinic pesticides50Rate of cross reaction with
NI means that no inhibition was observed in 50. mu.g/mL of analyte
Example 5 detection of thiamethoxam in food products
Accurately weighing two equal parts of 20.0g apple and tomato samples into a 80mL centrifuge tube, adding 40mL acetonitrile, homogenizing and extracting for 1min at 15000r/min by a high-speed tissue triturator, adding 5g sodium chloride, homogenizing and extracting for 1min, centrifuging for 5min at 3800r/min, taking 20mL (equivalent to 10g sample amount) of supernatant, carrying out rotary concentration in a 40 ℃ water bath to about 0.5mL, and placing the concentrate on a nitrogen blow-drying instrument for blow-drying.
3)1 part is used for ic-ELISA detection, the used solvent is 10mL PBS, 1 part is used for high performance liquid chromatography-tandem mass spectrometry detection, and the used solvent is 1mL acetonitrile + water (3+ 2); the apple samples are added with thiamethoxam at the concentration of 20 ng/mL, the apple samples are added with thiamethoxam at the concentration of 30ng/mL, and the tomato samples are added with thiamethoxam at the concentration of 15ng/mL, the tomato samples are recovered and detected, and the table 4 shows. The result shows that the recovery rate of the ic-ELISA method established by the antibody obtained by immunizing a mouse with the complete antigen prepared by the method is 75-120% for detecting thiamethoxam in apples and tomatoes, and the recovery rate is 77-110% verified by high performance liquid tandem mass spectrometry LC-MS-MS, so that the ic-ELISA method established by the antibody prepared by the antigen has good practicability.
TABLE 4 test for recovery of thiamethoxam in apple and tomato
Claims (10)
1. The structural formula of the thiamethoxam hapten is shown as a formula I:
2. a method of preparing a thiamethoxam hapten as shown in formula I in claim 1, comprising the steps of: under the alkaline condition, the thiamethoxam reacts with 4-mercaptobenzoic acid to obtain the thiamethoxam hapten.
3. The method of claim 2, wherein: the alkaline condition can be provided by sodium hydroxide, and the molar ratio of the thiamethoxam to the sodium hydroxide is 1: 2-3;
the molar ratio of the thiamethoxam to the 4-mercaptobenzoic acid is 1:1.1-1: 1.5;
the reaction is carried out in a solvent, wherein the solvent is N, N-dimethylformamide;
the reaction is carried out under the condition of heating reflux, the temperature of the reaction is 110 ℃, and the time is 3-4 hours.
4. The production method according to claim 3, characterized in that: the method further comprises the steps of: after the reaction is finished, cooling the reaction system to room temperature, adding water to quench the reaction system, adjusting the pH value of the reaction system to be neutral, adding ethyl acetate to stratify, collecting an organic layer, drying and concentrating to obtain a thiamethoxam hapten crude product;
the method further comprises the step of purifying the obtained thiamethoxam hapten crude product, and the method comprises the following specific steps: dissolving the crude thiamethoxam hapten product with ethyl acetate again, adding 100-200 silica gel, uniformly stirring, concentrating, removing the solvent, and then carrying out column chromatography separation to obtain a purified thiamethoxam hapten; wherein the eluent for the column chromatographic separation is a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 3: 1.
5. A thiamethoxam complete antigen obtained by coupling the thiamethoxam hapten as claimed in claim 1 with a carrier protein.
6. A method for preparing the thiamethoxam complete antigen of claim 5, comprising the steps of:
a) dissolving thiamethoxam hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature to obtain solution A;
b) dissolving carrier protein in carbonate buffer solution to obtain solution B;
c) mixing the solution A and the solution B and stirring to obtain solution C;
d) putting the solution C into a dialysis bag, dialyzing the solution C in a phosphate buffer solution, and collecting the solution in the dialysis bag to obtain the thiamethoxam complete antigen;
preferably, in the step a), the dosage ratio of the thiamethoxam hapten to the dimethylformamide to the dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 20 mu mol: 1-2 mL: 60-120 mu mol; the stirring time is 18-24 hours;
in the step b), the carrier protein is bovine serum albumin specifically;
the dosage ratio of the carrier protein to the carbonate buffer solution is 0.4 mu mol: 3-6 mL;
in the step c), the stirring conditions are as follows: stirring for 10-12 hours at 4-25 ℃;
in the step d), the dialysis is performed for 6 times by changing the solution every 3 hours.
7. Use of the thiamethoxam hapten as defined in claim 1 or the thiamethoxam complete antigen as defined in claim 5 for the preparation of a thiamethoxam specific antibody.
8. A thiamethoxam-specific antibody prepared from the thiamethoxam complete antigen of claim 5.
9. Use of a thiamethoxam-specific antibody according to claim 8 in at least one of the following aspects:
(1) the application in the detection of thiamethoxam;
(2) the application in preparing the kit for ELISA detection of thiamethoxam;
(3) the application in preparing the colloidal gold test strip of thiamethoxam.
10. An enzyme-linked immunosorbent assay or kit for detecting thiamethoxam, comprising the thiamethoxam complete antigen of claim 5 and the thiamethoxam specific antibody of claim 8.
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