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A kind of immunoassay kit for detecting ethopabate, preparation method and application

A technology of ethoxybenzamide and methyl ethoxybenzoate is applied in the field of preparation and detection of immunoassay kits for ethoxybenzamide, can solve problems such as affecting drug monitoring, and achieves improved fusion efficiency and effect , the effect of enhancing stability, improving affinity and sensitivity

Active Publication Date: 2019-01-04
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no immunological analysis methods for ethopabate residues reported at home and abroad, which largely affects the monitoring of the drug in edible animal tissues

Method used

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  • A kind of immunoassay kit for detecting ethopabate, preparation method and application
  • A kind of immunoassay kit for detecting ethopabate, preparation method and application
  • A kind of immunoassay kit for detecting ethopabate, preparation method and application

Examples

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preparation example Construction

[0032] A preparation method of an immunoassay kit for detecting ethopabate, the steps are as follows:

[0033] Step 1, the preparation of ethopabate hapten

[0034] The ethopabate hapten refers to methyl 4-amino-2-ethoxybenzoate, which can combine with the corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate the animal body to produce antibodies alone. The synthesis method of the hapten can be prepared through the following two ways.

[0035] Route 1: Using p-aminosalicylic acid as a raw material, undergo two-step reactions of methyl esterification and ethylation to prepare methyl 4-amino-2-ethoxybenzoate. The specific steps are: first react p-aminosalicylic acid, methanol and concentrated sulfuric acid at 55°C-75°C for 5-24 hours to generate 4-amino-2-hydroxybenzoic acid methyl ester; then, take p-aminosalicylic acid Add methyl ester, diethyl sulfate, sodium carbonate and N,N-dimethylformamide into a microwave reactor, heat up to 50°C-100°C, ...

Embodiment 1

[0072] Example 1 A preparation method of an immunoassay kit for detecting ethopabate

[0073] Preparation of ethopabate hapten: using p-aminosalicylic acid as raw material, react 5g of p-aminosalicylic acid, 50mL of methanol and 200mL of concentrated sulfuric acid at 65°C for 16h to generate 4-amino-2-hydroxybenzoic acid Methyl ester: Take 2g of methyl p-aminosalicylate, 3g of diethyl sulfate, 10g of sodium carbonate and N,N-dimethylformamide into a microwave reactor, heat up to 70°C, microwave power is 5W, and react for 2h . Cool the reaction kettle to room temperature, take out the product, add water, extract twice with dichloromethane, combine the extracts, and distill under reduced pressure to obtain 4-amino-2-ethoxybenzoic acid methyl ester, namely ethopabate half antigen.

[0074] Preparation of ethopabate immunogen and coater: dissolve 20mg polylysine in 30mL 0.01mol / L pH 7.4 phosphate buffer, add 7mL 25% glutaraldehyde solution, react at room temperature 18h, after ...

Embodiment 2

[0086] Example 2 Ethoxylate immunoassay kit detects ethopabate in chicken

[0087] Weigh 5g of fresh chicken tissue sample, cut it into pieces, grind it, mix it with phosphate buffer solution with pH 7.4, add 50mmol / L hydrochloric acid solution, mix it, and extract it by ultrasonic wave for 1h. After cooling to room temperature, filter, take 10 mL of the filtrate, add 1.0 mL of 1 mol / L sodium hydroxide solution, mix well, add 5.0 mL of 0.50 mol / L potassium dihydrogen phosphate solution, place at 4°C for 1 hour, filter, and take the filtrate 5.0 mL was added to a C18 solid-phase extraction cartridge, the sample was eluted with 3.0 mL methanol, the eluate was evaporated to dryness in a water bath, and dissolved in 1.0 mL distilled water for detection.

[0088] During detection, take out the kit prepared in Example 1, take out the microplate as required after returning to room temperature, add 50 μ L / well of ethopabate standard solution or sample solution to be tested in the micr...

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Abstract

The invention relates to an immunoassay kit for detecting ethopabate, and a preparation method and application thereof, belonging to the field of immunological detection. The kit comprises an ELISA plate coated with an ethopabate antigen, an ethopabate monoclonal antibody solution, an enzyme-labeled or fluorescently-labeled goat anti-mouse secondary antibody, a standard ethopabate solution, a washing solution and a diluent, wherein the ethopabate antigen is a conjugate of ethopabate semiantigen and a vector, and the vector is one or two selected from a group consisting of ovalbumin, bovine serum albumin, thyroid protein, hemocyanin, human serum albumin and polylysine. When applied to detection of ethopabate residues in animal food, the kit has the advantages of high sensitivity, precision and accuracy, good specificity, low cross-reaction rate, good stability, long storage time, easy operation, quickness and suitability for on-site primary screening of large quantities of samples, and is beneficial for simplifying residue analysis of ethopabate.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to an immunological kit for detecting ethopabate, a preparation method and an application thereof. Background technique [0002] Ethoxyabate is a broad-spectrum anticoccidial synergist with the chemical name methyl 4-acetamido-2-ethoxybenzoate, which inhibits shedding of infected Eimeria maxima eggs from chickens Capsules, block the synthesis of tetrahydrofolate in the p-aminobenzoic acid-folate metabolic pathway to exert anticoccidial effect. In veterinary clinics, the drug is used in combination with certain coccidiostats to produce a synergistic effect. It is used in poultry feed as a synergist of anticoccidials. It is often used in combination with amprolidine and sulfaquinoxaline to control Coccidiosis of poultry. The drug does not undergo metabolism in the poultry body, and is eliminated and excreted in the form of the drug. [0003] The Ministry of Agricu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/549G01N33/577G01N33/533G01N33/535G01N33/53
CPCG01N33/533G01N33/535G01N33/544G01N33/577G01N33/9493G01N2333/455
Inventor 李兆周陈秀金王耀樊振江高红丽李道敏李松彪曹力吕璞金东亮张为民
Owner HENAN UNIV OF SCI & TECH
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