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Nitrofurans medicament metabolite residue ELISA kit and use method

An enzyme-linked immunosorbent assay, an enzyme-linked immunosorbent assay technology, applied in the field of enzyme-linked immunosorbent assay, can solve the problems of prolonged detection time, difficult to apply in practice, single drug metabolite residue detection, etc.

Active Publication Date: 2008-08-13
SOUTH CHINA AGRI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] At present, there are immunoassay techniques and kits for the detection of metabolites of nitrofuran antibiotics, but all of them can only detect single drug metabolite residues. , and searching for relevant domestic patents are all single drug metabolite residue detection kits and methods, such as the patent "Enzyme-linked immunoassay kit for detecting nitrofurazone metabolites and its application", application number: 200710063871.8; patent "Nitrofurantoin metabolite enzyme-linked Immunoassay kit and its application", application number: 200710063837.7; patent "enzyme-linked immunoassay kit for detecting furaltadone metabolites and its application", application number: 200710063872.2; patent "an enzyme-linked immunoassay kit for furazolidone residue analysis Immunoassay Kit and Application", application number: 200510086346.9, etc.
However, as a high-throughput primary screening method, immunoassay technology is of little significance if it can only detect a single metabolite, especially for the detection of metabolite residues of nitrofuran antibiotics, because nitrofuran antibiotics include several species, such as furazolidone, furaltadone, nitrofurazone, nitrofurantoin, etc., and their metabolites are 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazole alkanone (AMOZ), semicarbazide (SC) and 1-amino-hydantoin (AH), so the detection unit needs to detect these types of metabolites in actual detection. If the immune kit can only detect one Because there are 4 metabolites, one sample must be tested 4 times, which will prolong the detection time, increase the detection workload, and increase the detection cost, and lose the significance of the primary screening method
[0008] In short, the existing nitrofuran immunoassay methods at home and abroad have a single detection object, which is difficult to apply in practice, and the existing products generally have shortcomings such as poor stability, complicated sample pretreatment and detection steps, high equipment requirements, and high prices. , has seriously affected the detection and monitoring of nitrofuran antibiotics. Therefore, it is of great economic and social significance to develop a multi-residue ELISA kit for multi-residue detection, simple operation, low equipment requirements, and cheap nitrofuran antibiotic metabolites.

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  • Nitrofurans medicament metabolite residue ELISA kit and use method

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Experimental program
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Embodiment 1

[0096] The preparation of embodiment 1 antigen

[0097] (1) Preparation of nitrofuran metabolite hapten:

[0098] a. Synthesis of metabolite AOZ hapten

[0099] 3-Amino-2-oxazolidinone (AOZ) reacts with p-aldehyde benzoic acid to form a hapten with a carboxyl group.

[0100] b. Synthesis of metabolite AMOZ hapten

[0101] 3-Amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) reacts with p-aldehyde benzoic acid in water to generate a hapten with a carboxyl group.

[0102] c. Synthesis of metabolite SC hapten

[0103] Reduce the nitro group of semicarbazide (SC) to amino group to generate hapten with amino group.

[0104] d. Synthesis of metabolite AH hapten

[0105] Amino-hydantoin (AH) reacts with m-aldobenzoic acid in water to form a carboxyl-bearing hapten.

[0106] (2) Preparation of nitrofuran metabolite antigen:

[0107] a. Dissolve 50 μmol / L nitrofuran metabolite hapten in 1 mL of DMF, then add equimolar DCC and NHS to the solution, let it react overnight at room temp...

Embodiment 2

[0111] The preparation of embodiment 2 antibody

[0112] Preparation of mouse monoclonal antibody against nitrofuran metabolites:

[0113] Animal immunization procedure: Balb / c mice were used as immunized animals, nitrofuran metabolite hapten and bovine serum albumin conjugate were used as immunogen, and the immunization dose was 60 μg / mouse. Freund's complete adjuvant was mixed to make an emulsifier, injected intraperitoneally, and the same dose of immunogen plus an equal amount of Freund's incomplete adjuvant was mixed and emulsified at intervals of 3 weeks, and the booster was given once. splenocytes.

[0114] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 4:1. Cell supernatants were measured by indirect competitive enzyme-linked immunosorbent assay, and positive wells were screened. The positive wells were cloned by microcloning until a hybridoma cell line stably secreting the monoclonal antibody was obta...

Embodiment 3

[0117] The extraction of embodiment 3 horseradish peroxidase or alkaline phosphatase

[0118] 1. Extraction of horseradish peroxidase

[0119] a. Water extraction: take by weighing 20 kilograms of washed fresh horseradish or horseradish skin, cut into small pieces, and mince in a pulverizer. Add 10 kg of water to the crushed slag slurry, stir and extract at low temperature for 8 hours, centrifuge at 3000 rpm for 10 minutes, and collect the supernatant.

[0120] b. Fractional separation of ammonium sulfate: add 226 grams of ammonium sulfate powder per liter of filtrate, stir while adding, put overnight at room temperature. The next day, draw the supernatant, and then add 258 grams of ammonium sulfate powder per liter of supernatant, and stir as you add it. After the ammonium sulfate is completely dissolved, put it in a cold room overnight. The next day, the supernatant was sucked off, and the precipitated part was centrifuged at 13,000 rpm for 20 minutes in a refrigerated cen...

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Abstract

The present invention discloses enzyme-linked immune kits for detecting nitrofurans metabolite residue including antigenic enzyme label plate coated with nitrofuran meatbolites, enzyme labeled antibody working fluid, standard solution, substrate solution, substrate buffer solution, reaction termination liquid, condense washing liquid and sample diluted concentrated liquid. The present invention also discloses using method for detecting by said kits including steps of pretreatment of sample, detecting with kits, result process and analysis and so on. The kits provided by present invention employ directly competing enzyme-linked immunoadsorption analysis to detect not only multiple nitrofurans metabolite residues at the same time, but also singleness nitrofurans metabolite residue, avoid many times detection of the same sample and reduce miss ratio, has merits of high sensitivity, good stability, less steps and low cost, and suit for screening of a large mount of samples, and has importance practical significance.

Description

technical field [0001] The invention relates to the technical field of enzyme-linked immunoassay detection, in particular to an enzyme-linked immunoassay kit for detecting residues of nitrofuran drug metabolites in animal-derived foods and a method thereof. Background technique [0002] Nitrofurans are synthetic broad-spectrum antibacterial drugs with the basic structure of 5-nitrofuran. Nitrofuran antibiotics mainly include Furazolidone, Furaltadone, Nitrofurazone, Nitrofurantoin, etc., are widely used in the prevention and treatment of animal infectious diseases such as poultry, livestock, aquatic products, and bees because of their antibacterial and bactericidal properties. Some species have growth-promoting effects and can be used as feed additive. [0003] Nitrofurans are a class of potentially carcinogenic and mutagenic substances. Due to the rapid metabolism of nitrofuran antibiotics such as furazolidone, furaltadone, nitrofurazone, and nitrofurantoin in the body, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/535G01N33/577
Inventor 沈玉栋杨金易孙远明肖治理雷红涛王弘
Owner SOUTH CHINA AGRI UNIV
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