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Enzyme linked immuno kit for detecting residual phenylethanolamine A and application method thereof

An enzyme-linked immunosorbent reagent and phenylethanolamine technology, which can be used in measurement devices, color/spectral property measurement, instruments, etc., and can solve the problems of complicated operation, difficult promotion, and expensive equipment.

Active Publication Date: 2013-01-30
JIANGSU WISE SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The prior art mainly uses high-performance liquid chromatography-tandem mass spectrometry, high-performance liquid chromatography and other detection methods for the detection of phenylethanolamine A, but the detection process of these methods is loaded down with trivial details, the detection time is long, the equipment needed is expensive, the operation is complicated, and it is difficult to to promote

Method used

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  • Enzyme linked immuno kit for detecting residual phenylethanolamine A and application method thereof
  • Enzyme linked immuno kit for detecting residual phenylethanolamine A and application method thereof
  • Enzyme linked immuno kit for detecting residual phenylethanolamine A and application method thereof

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Embodiment approach

[0052] According to a preferred embodiment of the present invention, the operation steps of the method of the present invention are:

[0053] Take a 96-well plate coated with phenylethanolamine A antigen, add 50 mL / well of phenylethanolamine A standard and processed samples to the corresponding microwells; add 50 mL / well of phenylethanolamine A antibody working solution, gently Shake and mix well, cover the plate with a cover film, and place it to react in a dark environment at 25°C for 30 min; carefully remove the cover film, spin dry the liquid in the well, and wash with 250 mL / well of washing working solution for 4~5 minutes. 10 s between each time, pat dry with absorbent paper; add 100 mL / well of phenylethanolamine A enzyme-labeled secondary antibody, gently shake and mix, cover the plate with a cover film, and place it at 25 °C for 30 min in a dark environment. , take out and repeat the plate washing as above; add 100 mL / well of substrate solution, gently shake and mix, c...

Embodiment 1

[0070] Example 1 Preparation of PEA-ELISA kit

[0071] PEA is a small molecule hapten, which has only reactogenicity but no immunogenicity. It needs to be combined with macromolecular substances before it can be used to immunize animals to prepare antibodies.

[0072] Phenylethanolamine A (PEA) drugs belong to small molecular substances and are haptens, which have only reactogenicity but no immunogenicity. Take phenylethanolamine A, modify the amino active group in its molecular structure, and couple with carrier protein to prepare a public antigen against phenylethanolamine A. In this study, phenylethanolamine A was used as hapten and carrier protein (BSA, etc.) to prepare artificial antigen by carbodiimide method.

[0073] Preparation of PEA-BSA antigen:

[0074] Mix PEA hapten and carrier protein BSA in 0.05 mol / L carbonate buffer (CBS) pH 9.6 at a binding ratio of 11:1, then add carbodiimide, stir for 1 to 2 hours, and leave to react at room temperature 24h, and finally...

Embodiment 2

[0112] Embodiment 2 adds phenylethanolamine A and reclaims

[0113] 1 ppm of PEA stock solution was diluted to an appropriate concentration and added to urine or pork samples identified by high performance liquid chromatography (HPLC) with no PEA residues at final concentrations of 0.2 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 4.0 ng / mL, 5 replicates of each concentration of samples were used for additive recovery experiments.

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Abstract

The invention discloses an enzyme linked immuno kit for detecting residual phenylethanolamine A and an application method thereof and belongs to the technical field of enzyme-linked immunosorbent assay methods. The enzyme linked immuno kit comprises an elisa plate coated by a benzene ethanolamine A antigen, phenylethanolamine A standard substance working solution, phenylethanolamine A antibody working solution, phenylethanolamine A IgG-HRP working solution, substrate solution, stop solution, concentrated washing solution and concentrated sample diluent. The enzyme linked immuno kit adopts an indirect competition enzyme-linked immunosorbent assay (ELISA) method. Residual phenylethanolamine A on the standard substance or the sample to be detected and an antigen pre-coated on the elisa plate contend for the phenylethanolamine A antibodies together. The application method can be used for directly detecting the phenylethanolamine A in animal derived food, urine samples, animal tissues and serum samples. The enzyme linked immuno kit has the advantages of being convenient, fast, sensitive and the like, and is suitable for detection of large-volume samples. In addition, the sensitivity of the enzyme linked immuno kit is 0.1ng / mL.

Description

[0001] technical field [0002] The invention discloses an enzyme-linked immunosorbent assay kit for detecting phenylethanolamine A in animal tissues and its metabolites and a detection method thereof, belonging to the technical field of enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA). The invention relates to an enzyme-linked immunoimmunoassay kit for detecting the content of residual phenylethanolamine A in animal source food, blood and urine and a detection method using the kit. Background technique [0003] Phenylethylamine A (Phenylethylamine, PEA), also known as Clonbamine, scientific name is 2-[4-(4-nitrophenyl)butyl-2-ylamino]-1-methoxyphenylethanol, is a A synthetic chemical substance. The chemical structural formula is as follows: [0004] [0005] Clombamine is an isomer of formoterol, a by-product of the synthesis of ractopamine, has the same action and effect as clenbuterol and ractopamine, and belongs to a kind of β-agonist, wh...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N21/31
Inventor 杜道林洪霞张祯薛永来
Owner JIANGSU WISE SCI & TECH DEV
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