Preparation Of Monoclonal Antibody Against Difloxacin And Development Of Colloidal Gold Test Strip

Fluoroquinolones are therapeutic drugs for bacterial infectious diseases.They have the characteristics of strong antibacterial ability,high efficiency,low cost,low cross-resistance,simple structure and strong tissue permeability.However,excessive and over-range use in the production and breeding process leads to drug resistance of some pathogenic bacteria and reduces the therapeutic effect.At the same time,the residual fluoroquinolones also have certain harm and toxicity to the central nervous system and liver and kidney organs of the human body,so it is necessary to establish a rapid and sensitive detection method.In this experiment,a method for rapid detection of difloxacin residues in animal-derived foods was established by combining monoclonal antibody preparation with colloidal gold immunochromatography.（1）The only immunogenic difloxacin was combined with the protein to form an artificial antigen.The concentrations of the immune antigen and the coating antigen were 2.5 mg·m L^-1 and 1.8 mg·m L^-1.The protein concentration of the synthesis was 2.5 mg·m L^-1 and 1.8 mg·m L^-1,respectively.The mice were immunized by abdominal administration.Through serum screening,No.4 mice with higher serum titer were finally selected,and their spleens were taken for cell fusion.（2）Culture myeloma cells and mouse macrophages in advance to confirm that cell fusion can proceed smoothly.Myeloma cells SP2/0 were fused with mouse splenocytes in a ratio of 1:5,and 30 positive wells were obtained after initial screening.After rescreening,four clonal cells were selected to enter the subclonal stage,and finally hybridoma cell lines 4A11-G2-C12-A10 were selected to be injected into mice for the preparation of ascites.（3）Immunochromatographic test strips were prepared using colloidal gold as a signal output carrier.The colloidal gold was observed and judged by visual observation and transmission electron microscopy（TEM）to determine the optimal conditions for the combination of colloidal gold and antibody.The sensitivity,cross-reaction,stability and sample addition test of the prepared colloidal gold test strip for difloxacin showed that the test strip was sensitive and stable,and could be used for the detection of DIF in food.In summary,this study established a method for detecting difloxacin residues in milk,which can be applied to the rapid detection of milk products.This method has broad application prospects and is expected to be applied to other food safety on-site screening.