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Kit for detecting zilpaterol by direct competitive ELISA (enzyme-linked immunosorbent assay) method

A technology of enzyme-linked immunosorbent reagents and immune reagents, which is applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of complex instruments and equipment, cumbersome detection process, and not suitable for screening a large number of samples, so as to reduce operation steps, The effect of low pretreatment requirements and no radioactive isotope pollution

Active Publication Date: 2012-12-12
浙江迪恩生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing zilpaterol detection method is chromatography, but these methods require a complete laboratory, experienced technicians, complex instruments and equipment, and the detection process is cumbersome, which is not suitable for the screening of a large number of samples on site

Method used

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  • Kit for detecting zilpaterol by direct competitive ELISA (enzyme-linked immunosorbent assay) method
  • Kit for detecting zilpaterol by direct competitive ELISA (enzyme-linked immunosorbent assay) method
  • Kit for detecting zilpaterol by direct competitive ELISA (enzyme-linked immunosorbent assay) method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Synthesis of Zipaterol Immunogen and Preparation of Immune Serum

[0022] 1.1 Reagents and instruments

[0023] Zipaterol (Beijing Bailingwei Reagent Co., Ltd.), succinic anhydride (Sigma Company), pyridine (Sigma Company), N, N-dimethylformamide (Dimethylformamide, DMF, Sigma Company), n-tributylamine (Tributylamine, Sigma Company) company), isobutyl chloroformate (Sigma Company), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH, Sigma Company), horseradish peroxidase (HRP), etc. (Shanghai Kaiyang Biotechnology Co., Ltd.), and other reagents were of analytical grade.

[0024] Double-beam ultraviolet-visible spectrophotometer (TU-1909, Beijing Puxi General Instrument Co., Ltd.), chromatography device (3057 portable recorder, Chongqing Chuanyi No. 4 Factory; SBS series numerical control drop counter, constant flow pump, automatic part Collector, chromatography column, Shanghai Huxi Analytical Instrument Factory), magnetic stirrer (Shanghai Dongrongf...

Embodiment 2

[0052] Embodiment 2 Establishment of direct competitive immunoassay method

[0053] 2.1 Checkerboard method to determine the optimal reaction concentration of ELISA

[0054] Coat the microtiter plate with 100 μl per well of zilpaterol antibody at a serial concentration of 1000 μg / ml, 100 μg / ml, 10 μg / ml, 5 μg / ml, 1 μg / ml, and 0.25 μg / ml, coat overnight at 4°C, and wash 3 times, pat dry, block overnight at 4°C with 200 μl blocking solution per well, wash 3 times, pat dry. Add enzyme-labeled zilpaterol diluted from 1:200, react at room temperature for 30 minutes, wash three times, add 100 μl of chromogenic reagent, protect from light at room temperature for 15 minutes, stop the reaction with 50 μl of stop solution, and detect the A value ( 450nm). Set two parallels at the same time, and take the coating concentration when the OD value is about 1.5 as the optimal concentration. The test data are listed in Table-4.

[0055] Table-4 Optimum Reaction Concentration Result Table ...

Embodiment 3

[0063] Embodiment 3 detects the formation of the ELISA kit of zilpaterol

[0064] An enzyme-linked immunosorbent assay kit for detecting zilpaterol was constructed to include the following components:

[0065] (1) Enzyme plate coated with zilpaterol antibody;

[0066] (2) Zipaterol horseradish peroxidase marker working solution;

[0067] (3) 6 bottles of zilpaterol standard solution, the concentrations are 0μg / L, 0.15μg / L, 0.3μg / L, 0.6μg / L, 1.25μg / L, 2.5μg / L, 5.0μg / L, 10μg / L;

[0068] (4) Mixed chromogen, including substrate hydrogen peroxide or carbamide peroxide, hydrogen donor tetramethylbenzidine or o-phenylenediamine;

[0069] (6) The washing liquid is a phosphate buffer containing 0.05% Tween 20;

[0070] (7) The concentrated sample diluent is a phosphate buffered saline solution of 0.1% Tween-20;

[0071] (9) The stop solution is 2mol / L sulfuric acid solution.

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Abstract

The invention discloses a kit for detecting zilpaterol by the direct competitive ELISA (enzyme-linked immunosorbent assay) method. The kit provided by the invention comprises a zilpaterol specific antibody, an ELISA plate coated with the zilpaterol specific antibody, zilpaterol standard, a zilpaterol HRP (horse radish peroxidase) label and a working solution. The ELISA kit for detecting zipaterolis sensitive, high-rate and accurate, so that the ELISA kit is mainly used for screening large quantities of samples; the major reagent in the kit is provided in the form of working solutions, so thatthe application is convenient, and the kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like; and the kit can rapidly detect the residual zilpaterol in feed and animal products.

Description

technical field [0001] The invention belongs to the technical field of ELISA and veterinary drug residue detection. Specifically, the invention relates to a direct competition ELISA kit for detecting zilpaterol. Background technique [0002] Zipaterol (Melamine), chemical name is (+ / 1)-trans-4,5,6,7-tetrahydro-7-hydroxy-6-(isopropylamino)imidazo[4,5, 1-jk]-[1]benzazepine-2(1H)-one, which is also a β-agonist, has the same physiological effect as clenbuterol (commonly known as "clenbuterol"), which can cause human or Animals suffer from heart failure, muscle tremors and, in severe cases, death. Zipaterol and other beta-stimulants are generally illegal. [0003] However, with the maturity and detection of drugs such as clenbuterol and ractopamine, as well as the intensification of efforts to combat them, similar drugs such as zilpaterol tend to be used by criminals. Therefore, it is necessary to establish a detection method for zilpaterol to prohibit the occurrence of such i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/52G01N21/31G01N33/535
Inventor 王旻子于洪侠张明洲程晔魏建良
Owner 浙江迪恩生物科技股份有限公司
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