Preparation methods and application of hapten and complete antigen for parathion

A complete antigen and parathion technology, applied in the biological field, can solve the problems of long time, high cost, high cost, etc., and achieve the effects of strong specificity, high sensitivity, and simple and easy preparation method.

Active Publication Date: 2020-05-19
GUANGXI SUBTROPICAL CROPS RES INST GUANGXI SUBTROPICAL AGRI PROD PROCESSING RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, chromatographic methods are widely used in the detection of parathion, including gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), supercritical Fluid chromatography (SFC), capillary electrophoresis (CE), gas chromatography-infrared spectroscopy (GC / FTLR) and other methods, but these methods require expensive instruments, professional operators, and sample pretreatment is complicated, costly, and time consuming Therefore, it is of great significance to establish a fast and simple method for the detection of parathion

Method used

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  • Preparation methods and application of hapten and complete antigen for parathion
  • Preparation methods and application of hapten and complete antigen for parathion
  • Preparation methods and application of hapten and complete antigen for parathion

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1, the preparation of parathion hapten

[0047] Include the following steps:

[0048] 1) Dissolve 1 part (mole parts, the same below) of methyl 3-chloro-4-hydroxybenzoate in 3-4 parts of acetonitrile, add 2-3 parts of anhydrous potassium carbonate and 1.2 parts of chlorothiophosphoric acid di Ethyl ester, the reaction system was heated to reflux at 80°C for 2 to 3 hours. After the reaction system was cooled to room temperature, 20 parts of water was added to quench the reaction, and then 20 parts of ethyl acetate was added to extract and separate the layers. The organic layer was collected, dried and concentrated, and then separated by column chromatography to obtain Intermediate: methyl 3-chloro-4-((diethoxythiothio)oxy)benzoate.

[0049] 2) Dissolve 1 part of 3-chloro-4-((diethoxythiothio)oxy)methyl benzoate in 3-4 parts of tetrahydrofuran, add an equal volume of 6mol / L hydrochloric acid, and stir the reaction system at room temperature After 12 hours, ad...

Embodiment 2

[0051] Prepare parathion complete antigen with the parathion hapten of embodiment 1, comprise the following steps:

[0052] 1) Dissolving the organophosphorus pesticide hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature for 18 hours to obtain liquid A; wherein, the organophosphorus pesticide hapten, The consumption ratio of dimethylformamide, dicyclohexylcarbodiimide and N-hydroxysuccinimide is 20 μmol: 1mL: 60 μmol: 60 μmol;

[0053] 2) dissolving the carrier protein in carbonate buffer solution to obtain liquid B; wherein, the dosage ratio of carrier protein and carbonate buffer solution is 0.4 μmol: 3 mL;

[0054] 3) Add the above liquid A dropwise to liquid B and stir at 4°C for 10 hours to obtain liquid C;

[0055] 4) Put liquid C in a dialysis bag and dialyze it in phosphate buffer, change the solution every 3 hours, dialyze 6 times in total, collect the solution in the dialysis bag, which is the complet...

Embodiment 3

[0058] Identification of parathion complete antigen.

[0059] The Bal b / c mice were immunized with the complete parathion antigen prepared in Example 2, and the orbital vein serum was collected on the 5th day after the fourth immunization to detect whether the prepared parathion antigen had immune activity.

[0060] 1) The parathion complete antigen (1mg / mL) prepared in Example 2 was diluted by 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000 times and added to the microtiter plate, see Table 1, 100uL per hole, After incubating at 37°C for 3 hours, take it out and wash it with PBST;

[0061] 2) Add 50uL of PBS to well 0 of the coated enzyme-labeled well, add 50uL of parathion standard sample with a concentration of 100ng / L to the inhibition well; take the collected mouse serum, dilute 500, 1000, 2000, 4000, 8000 , 16000 times into the enzyme-labeled wells, 50uL per well, see Table 1, incubate the enzyme-labeled plate at 37°C for 0.5h, take it out, and wash it with PBST;

[0...

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Abstract

The invention discloses preparation methods and application of a hapten for parathion and a complete antigen for parathion. The hapten for parathion is prepared by dissolving 1 part of 3-chloro-4-methyl hydroxybenzoate into 3-4 parts of acetonitrile; adding 2-3 parts of anhydrous potassium carbonate and 1.2 parts of diethyl chlorothiophosphate to prepare an intermediate, namely 3-chloro-4-((diethoxythio)oxy)methyl benzoate; dissolving the intermediate into 3-4 parts of tetrahydrofuran; and adding 6 mol / L of isometric hydrochloric acid to prepare a compound, namely 3-chloro-4-((diethoxythiophosphoryl)oxy)benzoic acid which is the hapten for parathion. The hapten is coupled with carrier protein to prepare the complete antigen. Immune animal experiments show that the artificial antigens prepared by the invention have good immunogenicity. The hapten and complete antigen provided by the invention can be used for immunoassay detection of parathion, and have very wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of parathion hapten and complete antigen. Background technique [0002] Organophosphorus pesticides have been widely used in my country due to their broad-spectrum and high-efficiency characteristics. According to relevant statistics, their output has accounted for almost 1 / 3 of my country's market share in the past 10 years. Research has found that organophosphorus pesticides used in large quantities fail to degrade in time, and have a longer half-life under certain conditions, and can accumulate in organisms through the food chain. and safe exits. Parathion is a kind of organophosphorus pesticide. Due to its high toxicity, carcinogenesis, teratogenicity and mutagenicity, parathion has been banned in my country and has been listed as a key monitoring object. my country's national food safety standard GB2763-2016 "Maximum Residue Lim...

Claims

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Application Information

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IPC IPC(8): C07F9/18C07K14/765C07K1/107C07K16/44G01N33/53
CPCC07F9/18C07K14/765C07K16/44G01N33/5308
Inventor 吕丽兰何丽珊张娅邹承武甘志勇彭靖茹吴静娜吕玉莲吴凤李鸿李乾坤杜国冬何惠欢易国强温绍聪陈能刘唯
Owner GUANGXI SUBTROPICAL CROPS RES INST GUANGXI SUBTROPICAL AGRI PROD PROCESSING RES INST
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