Method for detecting maduramicin and special enzyme-linked immunoassay reagent kit thereof
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An enzyme-linked immunosorbent reagent, maduramycin technology, applied in immunoglobulins, chemical instruments and methods, biochemical equipment and methods, etc. process, etc.
Inactive Publication Date: 2011-06-01
北京维德维康生物技术有限公司
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[0004] The current chemical methods for detecting the content of maduramycin include high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, which are not suitable for on-site monitoring and screening of a large number of samples due to complex equipment and cumbersome processes
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Embodiment 1
[0064] Embodiment 1, preparation of maduramycin hapten, maduramycin antigen and maduramycin monoclonal antibody
[0065] 1. Maduramycin hapten and its preparation
[0066] Maduramycin amine is purchased from Sigma-Aldrich Company, and the product catalog number is 34069-100MG;
[0067] Dissolve 100 mg of maduramycin amine in 1 mL of dioxane, stir to dissolve completely; then add 70 μL of anhydrous tri-n-butylamine, place the mixture in an ice bath at 4°C to cool, and stir for 15 min. Add 15 μL of re-distilled isobutyl chloroformate, stir at 4°C for 30 minutes to obtain solution I; weigh 30 mg of p-aminobenzoic acid and dissolve it in water, stir to dissolve, adjust the pH to 9.0 with 0.1mol / L NaOH, and obtain Solution II; solution I was added dropwise to solution II, and stirred slightly at 4° C. for 4 hours to obtain solution III, which contained the maduramycin hapten compound represented by formula I.
[0068]
[0069] 2. Maduramycin antigen and its preparation
[007...
Embodiment 2
[0103] Example 2, ELISA kit for detecting maduramycin and its preparation and application
[0104] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:
[0105] 1. Maduramycin coater;
[0106] 2. ELISA plate;
[0107] 3. Maduramycin monoclonal antibody: secreted by the maduramycin monoclonal antibody hybridoma cell line MAD with the deposit number CGMCC No.3391;
[0108] 4. Maduramycin standard substance: the concentration of the standard solution is 0μg / L, 0.5μg / L, 2μg / L, 8μg / L, 32μg / L and 128μg / L; the maduramycin standard substance is Maduramycin Prime amine (MADURAMICIN AMMONIUM), purchased from Sigma-Aldrich Company; Product catalog number is 34069-100MG; Diluted to above-mentioned each concentration with sample diluent;
[0109] 5. Goat anti-mouse secondary antibody labeled with horseradish peroxidase;
[0110] 6. Substrate chromogenic solution: composed of liquid A and liquid B, liquid A is an aqueous solution of 2% carbamide peroxide, li...
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Abstract
The invention discloses a method for detecting maduramicin and a special enzyme-linked immunoassay reagent kit thereof. The enzyme-linked immunoassay reagent kit comprises a maduramicin monoclonal antibody. The maduramicin monoclonal antibody is secreted by the hybridoma cell line MAD of the maduramicin monoclonal antibody, whose preservation number is CGMCC No. 3391. In the enzyme-linked immunoassay reagent kit of the invention, an indirect competition ELISA (Enzyme-Linked Immuno Sorbent Assembly) method is mainly used for qualitatively or quantitatively detecting the content of the maduramicin in animal muscles and livers. The reagent kit and the detection method of the invention have low requirements on the pretreatment of samples and simple pretreatment process of the samples and can detect mass samples quickly at the same time. By using the maduramicin monoclonal antibody with high specificity, the detection method is convenient and simple, and the invention has the characteristics of high specificity, high sensibility, high precision, high accuracy and the like.
Description
technical field [0001] The invention relates to a method for detecting maduramycin and a special ELISA kit. Background technique [0002] Melamine is a triazine nitrogen-containing heterocyclic organic compound, an important nitrogen-heterocyclic organic chemical raw material. Pure white monoclinic prism crystal, odorless. Soluble in hot water, slightly soluble in cold water, very slightly soluble in hot ethanol, insoluble in ether, benzene and carbon tetrachloride, soluble in methanol, formaldehyde, acetic acid, hot ethylene glycol, glycerin, pyridine, etc. It is generally stable, but it may decompose and release cyanide at high temperature. [0003] Since the nitrogen content in the molecular formula of maduramycin is about 66%, and the average nitrogen content of protein is about 16%, it is often used as a food additive by some unscrupulous businessmen to improve the protein content index in food testing. Therefore, maduramycin is commonly known as "protein essence". ...
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