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Rapid Determination of Chloramphenicol and Clenbuterol Residues in Food by the Competitive Fluorescence Microsphere Immunoassay

Author: GaoHaiXia
Tutor: JiangJiZhi;ZouMingQiang;QuZhanLiang
School: Hebei University
Course: Biochemistry and Molecular Biology
Keywords: chloramphenicol clenbuterol microsphere-based competitive fluorescent immunoassay microsphere flow cytometery
CLC: S859.84
Type: Master's thesis
Year: 2008
Downloads: 22
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Abstract


Chloramphenicol and clenbuterol hydrochloride have been widely utilized in order to pursue maximum productivity and profits directly or indirectly in food products. However, residues of such veterinary drugs in food products have been proven to be detrimental to human health. At present, chloramphenicol and clenbuterol hydrochloride have been already forbided to use, but some businessmans are still illicited use for pursuing economic benefits. Several methods have been developed to quantify such veterinary drugs, including GC, HPLC, MS, ELISA and FIA. However, each of these techniques possesses one or more significant limitations:GC, HPLC and MS are very expensive; ELISA will only measure one analyte as a time. The recent development of microsphere-based competitive fluorescent immunoassay has raised hopes that many of these limitations can be overcome.In this research, microsphere-based competitive fluorescent immunoassay was used, and combined with the principle of competitive immunoassay, in order to establish a high-throughput, rapid and sensitive method to determination of chloramphenicol and clenbuterol hydrochloride and other veterinary drugs in food.In this study, the hapten chloramphenicol and clenbuterol were linked to the carrier protein BSA by Diazo binding methods, and two kinds of coupled antigens were obtained. The successful coupling of the haptens and the BSA were proved by ultraviolet scan and mass spectrum. The incorporation ratio was 13 and 16, respectively. The successful labelling of FITC and antibody were proved by CAPillary electrophoresis and fluorospectrophotometer. And the signal of fluorescence probe can be detected by flow cytometery.It was the best that 10μg of CAP-BSA or 5μg of CL-BSA coupled to microspheres by optimization test. The optimal sensitivity was about 220ng/mL chloramphenicol monoclonal antibody and 70.5ng/mL clenbuterol monoclonal antibody. It has a sensitivity of 0.05ng/mL with dynamic range of 0.1-100ng/mL by determination of chloramphenicol. And it has a sensitivity of 0.005ng/mL with dynamic range of 0.01-100ng/mL by determination of clenbuterol. Detection sensitivity of microsphere-based competitive fluorescent immunoassay was 2 folds and 50 folds higher than that of ELISA for detection of chloramphenicol and clenbuterol. A total detection time of 3.5 h was needed including preparation of detection microspheres.Determination of chloramphenicol and clenbuterol residues in pig urines and in milk with this novel method, the recovery was in the range of 96%-119% in pig urines and in the range of 96%-102.4% in milk, respectively. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultra low trace sample consumption and low cost.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Pharmacology > Veterinary Toxicology > Chemical residues in animal foods
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