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

A technology of chemiluminescence enzyme and albuterol, which is applied in the direction of chemiluminescence/bioluminescence, analysis through chemical reaction of materials, measurement devices, etc., can solve the problems of complex processing, analysis of many interference factors, time-consuming, etc., and achieve high sensitivity 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 salbutamol
  • Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting salbutamol
  • Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting salbutamol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Preparation of immunogen, coated antigen and antibody

[0044] (1) Synthesis of immunogen

[0045] The immunogen was obtained by coupling salbutamol and bovine serum albumin (BSA) by para-aminobenzoic acid method. It includes the following steps:

[0046] A, Weigh 10mg (73μmol) of p-aminobenzoic acid and dissolve it in 1.1mL 0.2N HCl, and then weigh 6mg (87μmol) of NaNO 2 Dissolve in 0.35mL of distilled water, stir at 0-4℃, add NaNO 2The solution was added dropwise to the p-aminobenzoic acid solution and reacted for 1 hour in the dark to obtain solution A;

[0047] B. Weigh 10mg (42μmol) of salbutamol sulfate and dissolve it in 10mL of ice-cold 0.05M borax buffer (pH=8.5, containing 0.15M NaCl), stir at 0-4°C, add 0.9mL of the above A solution dropwise to In this solution, react in the dark for 2 hours to obtain an orange solution;

[0048] C, add a small amount of H to the solution 3 BO 3 Adjust the pH of the crystals to 7.4, then add 94mg (1.4μmol) cBSA (bovine...

Embodiment 2

[0058] Example 2. Establishment of SAL-ELISA detection method

[0059] (1) The optimal concentration of antibody and coating antigen (square matrix)

[0060] Longitudinal dilutions of each coated antigen at 80.0μg / mL, 40.0μg / mL, 20.0μg / mL, 10.0μg / mL, 5.0μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL Degree-coated microtiter plate, 100μL / well, place overnight at 0-4℃, wash the plate three times with washing solution, and pat dry each time; block with 250μL / well blocking solution, place at room temperature for 3 hours, wash the plate three times, pat dry each time ; Add 100μL / well of a series of diluted antibodies (1:100 to 1:1024000), place at room temperature for 2 hours, wash the plate three times, and pat dry each time; add 100μL / 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 to determine the luminescence value. The concentration of the...

Embodiment 3

[0072] Example 3. Chemiluminescence enzyme-linked immunoassay kit for detecting salbutamol

[0073] (1) Composition of chemiluminescence enzyme-linked immunosorbent assay kit for detecting salbutamol

[0074] A. Solid-phase carrier (enzyme-labeled plate) coated with coated antigen (conjugate of clenbuterol and carrier protein);

[0075] B. Salbutamol standard solution: 0.1ng / mL, 0.2ng / mL, 0.5ng / mL, 1ng / mL, 2ng / mL, 5ng / mL.

[0076] C. Enzyme-labeled goat anti-rabbit antibody solution: Enzyme-labeled goat anti-rabbit antibody is a stock solution of horseradish peroxidase- goat anti-rabbit IgG, which is loaded, and the washing solution is used to prepare a working concentration of 1:1000.

[0077] D. Salbutamol antibody solution: Polyclonal antibodies are prepared by immunizing animals with artificial immune antigens, and the resulting salbutamol antibody is diluted with a washing solution to a working concentration of 1:1000.

[0078] E. Luminous solution: Use 0.0001M p-cresol tris ...

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Abstract

The invention discloses a chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting salbutamol. The kit comprises a kit body, in which an ELISA plate with wells coated with coating antigen prepared from clenbuterol and ovalbumin by coupling, anti-ractopamine polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit antibody (enzyme-labeled secondary antibody), a series of salbutamol standard solutions, a concentrated phosphate buffer solution, a concentrated washing solution and a chemiluminescence solution are arranged. The CELISA kit has the advantages of high sensitivity, good repeatability, and good simplicity, rapidness and accuracy; 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 salbutamol in animal-derived foods (such as milk and animal tissues) and urine samples.

Description

Technical field [0001] The invention relates to an enzyme-linked immunoassay kit, in particular to a chemiluminescence enzyme-linked immunoassay kit for salbutamol. Background technique [0002] Salbutamol belongs to β-stimulant, β-stimulant is a kind of chemically synthesized phenylethanolamine derivatives. The mechanism of action of β-stimulant is the same as epinephrine and norepinephrine. It can affect the flow and redistribution of nutrients in animals, effectively promote muscle tissue growth, reduce carcass fat content, increase lean meat rate and increase lean meat production, So it has been widely used in Europe and America. Athletes use this medicine 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 promoters is generally higher, generally 5-10 times the therapeutic dosage. Facts have shown that...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/76
Inventor 郗日沫刘伟尹伟伟李伟华丁锴刘中秋
Owner SHANDONG UNIV
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