| Summary: | 碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 104 === Florfenicol (FF) is a broad-spectrum, as a class of amphenicol synthetic antibiotics, has been widely used in poultry, livestock and aquaculture. FF has reported to have male reproductive toxicity and a long-term consumption may cause testicular atrophy, decreasing sperm cells production and increasing the risk of leydig cell tumors. Ractopamine (Rac) is a β-adrenoceptor agonist drug that used in asthma medication, which can increase protein synthesis, thereby making the animal more muscular. Rac is widely used as a feed additive for livestock, reducing the fat content of the meat and increasing the profit per animal. Rac may cause some toxic symptom, such as tachycardia, vasodilation, skeletal muscle tremor, nervousness and metabolic disturbances. For these reasons, it is quite urgent to develop a way to detect the level of FF or Rac in our daily food. In this study, we have developed the sensitive and rapid methods for both these small compounds. Firstly, we derivatived the haptens by coupling FF or Rac with SH, and then confirmed the results of derivative by TLC. Polyclonal antibodies specific for FF are generated from the mice after being immunized with FF-SH-BSA. Using the antibody from the mice, sensitive cdELISA were developed. In the cdELISA of polyclonal antibody of FF, the concentration causing 50％ inhibition (IC50) of binding of Florfenicol-horseradish peroxidase (FF-HRP) to the antibody by FF is calculated to be 3.14 ng/mL. Monoclonal antibody specific for FF was also produced from a stable hybridoma cell line, 7G81A3, which was generated by the fusion of P3/NS1/1-AG4-1 myeloma cell with spleen cells isolated from Balb/c mouse. In the cdELISA of monoclonal antibody of FF, the IC50 of binding of FF-HRP to the antibody by FF is calculated to be 3.7 ng/mL. We also produced polyclonal antibody of Rac from the mice and the rabbits, respectively, by being immunized different Rac conjugate antigens. According to the result of cdELISA of Rac, the IC50 of binding of Rac-HRP to the mice or rabbit antibody by Rac are calculated to be 40.2 ng/mL and 20.4 ng/mL respectively. Following, the immunostrip for detecting FF and Rac were established, respectively. The detection limit of strip were 100 ng/mL and 1000 ng/mL for FF and Rac, respectively. Therefore, we have been successfully generated polyclonal antibody for Rac and monoclonal antibody for FF, and apply in ELISAs. However, due to the regulatory limit level of Rac set by Taiwan-FDA is 10 ng/mL. The immunostrip established in the present study is not sensitive enough for Rac analysis.
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