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Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine

A technology of ractopamine and chemiluminescent enzymes, which is applied in the fields of chemiluminescence/bioluminescence, analysis through chemical reactions of materials, and measurement devices, which can solve the problems of complex processing, high detection cost, and complicated operation process, and achieve high sensitivity high effect

Inactive Publication Date: 2009-06-03
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of microbiological methods are: time-consuming and lack of specificity
The disadvantages of thin-layer chromatography are: the operation process is complicated and takes a long time; the operators need to undergo professional training; there are many interference factors affecting the analysis, and the repeatability of the results is poor.
Thin-layer chromatography, radioimmunoassay, high-performance liquid chromatography, color / mass analysis, and liquid / mass analysis have the disadvantages of expensive instruments and equipment, complicated sample pretreatment, time-consuming, laborious, and difficult to popularize , the detection cost is high, especially the radioimmunoassay also needs to be equipped with radioactive sources, which has certain risks

Method used

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  • Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine
  • Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine
  • Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, preparation of immunogen, coated antigen and antibody

[0043] (1) Synthesis of immunogen

[0044] The immunogen was obtained by coupling ractopamine and bovine serum albumin (BSA) by the p-aminobenzoic acid method. Specifically include the following steps:

[0045] A. Weigh 14mg (100μmol) of p-aminobenzoic acid (ABA) and dissolve it in 1.5mL 0.2M hydrochloric acid, then weigh 8.3mg (120μmol) of sodium nitrite (NaNO 2 ) was dissolved in 0.24mL of distilled water, stirred at 0-4°C, sodium nitrite (NaNO 2) solution was added dropwise to the p-aminobenzoic acid solution, and reacted in the dark for 1 hour to obtain solution A;

[0046] B. Weigh 34mg (100μmol) of ractopamine and dissolve it in 5mL of ice-cold borax buffer (0.05M) (pH8.5, containing 0.15M NaCl), stir at 0-4°C, add 2mL of the above-mentioned A solution dropwise into the solution, and reacted in the dark for 2 hours to obtain an orange solution;

[0047] C. Add a small amount of boric acid c...

Embodiment 2

[0056] Embodiment 2, the establishment of CL-ELISA detection method

[0057] (1) Optimization of antibody and coated antigen concentration (square matrix)

[0058] Serially dilute each coated antigen longitudinally at 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3125 μg / mL Coat the microtiter plate with 100 μL / well, place overnight at 0-4°C, wash the plate three times with washing solution, and pat dry each time; block with 250 μL / well blocking solution, place at room temperature for 2 hours, wash the plate three times, and pat dry each time ; Add 100 μL / well of a series of diluted antibodies (1:100 to 1:1024000), place at room temperature for 2.5 hours, wash the plate three times, and pat dry each time; add 100 μL / well of 1:1000 horseradish peroxidase-sheep Anti-rabbit IgG antibody, place at room temperature for 1 hour, wash the plate three times, and pat dry each time; add 100 μL / well of luminescence solution, and measure the luminesc...

Embodiment 3

[0070] Example 3, Chemiluminescent ELISA Kit for Detection of Ractopamine

[0071] (1) The composition of the chemiluminescent ELISA kit for detecting ractopamine

[0072] A, coated with a solid phase carrier (enzyme plate) coated with antigen (conjugate of ractopamine and carrier protein);

[0073] B. Ractopamine standard solution: 0.01ng / mL, 0.05ng / mL, 0.1ng / mL, 1ng / mL, 5ng / mL, 10ng / mL.

[0074] C. Enzyme-labeled goat anti-rabbit antibody solution: Enzyme-labeled goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit IgG stock solution, loaded, and prepared with washing solution to a working concentration of 1:1000 when used.

[0075] D. Ractopamine antibody solution: use the polyclonal antibody prepared by immunizing animals with artificial immune antigens, and dilute the obtained ractopamine antibody with washing solution to a working concentration of 1:1000.

[0076] E, luminescent solution: use 0.0001M tris(hydroxymethyl)aminomethane solution of p-cresol ...

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Abstract

The invention discloses a chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine. The kit comprises a kit body, in which an ELISA plate with wells coated with coating antigen prepared from ractopamine and ovalbumin by coupling, a series of ractopamine standard solutions, enzyme-labeled goat anti-rabbit antibody, anti-ractopamine antibody, a chemiluminescence solution, a washing solution, a coating solution and a blocking solution are arranged. The CELISA kit is sensitive, simple, rapid and accurate; and the sensitivity is improved by one order of magnitude compared with that of the conventional colorimetric ELISA method. The CELISA kit can be used for detecting residual ractopamine in animal-derived foods (such as milk and animal tissues) and urine samples.

Description

technical field [0001] The invention relates to an ELISA detection kit, in particular to a ractopamine chemiluminescent ELISA detection kit. Background technique [0002] Ractopamine belongs to β-stimulants, and β-stimulants are a class of chemically synthesized phenylethanolamine derivatives. The mechanism of action of β-stimulants is the same as that of adrenaline and norepinephrine. It can affect the flow and redistribution of nutrients in animals, effectively promote the growth of muscle tissue, reduce carcass fat content, increase lean meat percentage and increase lean meat production. Therefore, it was widely used in Europe and America. Athletes use this drug to increase muscle and lung capacity and shorten the recovery period after high-intensity training. But on the other hand, β-stimulants have a chemical structure similar to adrenaline, and the dosage of such compounds as growth-promoting agents is generally higher, generally 5-10 times the therapeutic dosage. F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/76
Inventor 郗日沫尹伟伟刘伟李伟华丁锴刘中秋
Owner SHANDONG UNIV
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