Indirect Competitive ELISA For Detection Of Doxycycline Residues In Animal Edible Tissues

Doxycycline (DOX) is a synthetic antibiotics remain with the Tetracyclines groups. It has been widely used in veterinary medicine to cure microbial infections. However the DOX residues in animal edible tissues could cause serious Public healthy problems, so DOX residue in animal edible tissues has been one of the head monitored objects in veterinary drug residue study in a period of times. Many physical and chemical methods were study to detect residue of DOX and have been established at home and abroad. Some of them are precise and sensitive, but not suitable for large batch samples' screening; because of their verbose operate process, hight costs, and more technical request. Enzyme-linked immunosorbent assay (ELISA) is a expedient and fast method to screen large samples. At the same time, mainly drugs surveillant kits are depends on foreigns, so it is urgent to develope a drugs monitor ELISA fast kits for DOX with own independent property right. It is meaningful to establish a fast detection method to determine DOX residue to control animal food safety and safeguard consumers healthy.The purpose of the study was to develope an indirect competitive enzyme-linked Immunosorbent assay (ic-ELISA) to monitor DOX residues in animal edible tissue. The maincontents and result were as followed:1. Production and identification of Doxycycline polyclonal antibody. The immunogen and the coating antigen were synthesis. After immune the New Zealand rabbit, the antiserum titre and sensitivity were obtained using an indirect ELISA and ic-ELISA. The results showed:The antiserum titre of DOX was about 1:80,000 and the sensitivity was perfect.2. The optimal test of ic-ELISA method. The optimal of Doxycycline residues test conditions were as followed:The working consistence of coating antigen and polyclonal antibody were determined, which is 0.5μg/ml and 1:32,000 respectively. And the coating time was made at 4℃for overnight. The incubation time of antigen and antibody complex reaction was set in 15 min.3. The ic-ELISA method was established to detect Doxycycline residues. The standard curve was drawed by analysis of ic-ELISA, The IC50 was 8.74μg/L and the limit of detection was 1.96μg/L. The ic-ELISA has a dynamic range from 1.96 to 160μg/L The DOX polyclonal antibody developed here has greater affinity. The intra-assay average variation coefficients in the liver samples and the muscle samples were 5.75 and 7.53%, respectively. The inter-assay average variation coefficient was 5.92% with the liver samples and 7.21% for muscle sample. These results demonstrated that it was feasible to determine DOX using the ic-ELISA.4. The method was initially applied in the addition and reclamation experiment of the liver and muscle tissues of pigs. three kinds of DOX concentratin was added to liver and muscle samples to measure DOX recovery rate, and the recoveries of DOX in liver ranged from 80.19 to 89.41%. In muscle samples, the recoveries ranged from 83.98 to 94.75%.