Preparation Of Polyclonal Antibodies Agaist Tetracyclines And Studies On Amikacin Using ELISA Method

Tetracycline (TC) is a synthetic antibacterialt that belongs to the Tetracyclines group and Amikacin(Amk) is one of the most common semi-synthes penicillin that belongs to the Aminoglycoside group. They have been increasingly used in veterinary medicine to treat microbial infections. However, the TC and Amk residues in animal edible tissues could cause serious public health problems. Enzyme-linked immunosorbent assay(ELISA) is a convenient and fast method to screen large samples.But now, mainly kits depend on foreign, so it is urgent to develop ELISA fast kit for clenbuterol with own independent property right. It's very meaningful to establish a fast detection method to detect the two antibiotics residue to control animal food safety and safeguard consumers' health.The purpose of the study here is to develop an indirect competitive enryme-linked immunosorbent assays(Ci-ELISA) to monitor TC and Amk residues in animal food. The main contents and results were as followed:1. Synthesis and Identification of Tetracycline and Amikacin Artificial Antigen. The immunogen and the coating antigen were synthesized by carbodimide method. The complete antigens were identified by UV spectrometric scanning method and immune BALB/c mice tests. The results demonstrated that the artificial antigens were synthesized successfully.2.Synthesis and Identification of Tetracycline and Amikacin polyclonal antibody.The antiserum titre and specificity obtained using an indirect ELISA and Ci-ELISA respectively.The results showed: the antiserum titre of TC was about 1:80000 and the specificity was good; the antiserum titre of Amikacin was about 1:600000 and the specificity was good.3.The Ci-ELISA method was established to detect Amikacin residues.The optimal test conditions was as followed: coating antigen, blocking reagent , and horseradish peroxidase marked to the goat IgG of anti-rabbit IgG were 1:1000, 1% of separated milk, and 1:5000 respectively. Its standard curve is in 0.05μg/mL-1μg/mL, the curvy equation is y= 0.4641x-0.6958, coefficient correlation R= 0.9955, with a limit of detection to 40ng/mL, IC₅₀=0.38ng/mL. Precision of the Ci-ELISA indicated by coeffcients of variation of intra-assay and inter-assay were 19.7%, 12.7%,4.5% and 20.1%,9.5%,8.0%.There was no cross-activity between the same kind and five other kinds. Accuracy of the method denoted by mean recoveries of milk and kidney sample were 81%,78%,75% and 79%,76%,72%.4.Synthesis and Identification of Akacin monoclonal antibody were expounded in the research. The Ci-ELISA method was established to detect Amikacin residues. The curvy equation is y= 0.4942x-0.7554, coefficient correlation R= 0.9956, with a limit of detection to 44ng/mL, IC₅₀=0.35μg/mL. There was no cross-activity between the same kind and five other kinds. Accuracy of the method denoted by mean recoveries of milk and kidney sample was 84%, 79% and 72%.