Labeled Monoclonal Antibody Probe Of Tetracyclines By Colloidal Gold

Tetracyclines (TCs) have been widely used in stock raising and fishery. The improper usage of TCs leads to serious residues in edible animal foods. It is very important to establish a serious of quick, sensitive, accurate and convenient detection methods to monitor the TCs residues in animal foods.At present, the methods of HPLC, HPLC-MS, ELISA have been applied in the analysis of TCs residues. Compared with these methods, immunochromatographic assay of colloidal gold (GICA) presents rapid, sensitive and convenient properties. However, it is a first step that prepared colloidal gold labeled antibody probe in order to GICA.Therefore, the paper included: (1) to establish the hybridoma cell lines secreting monoclonal antibodies against tetracyclines; (2) to produce monoclonal antibody against tetracyclines; (3) to produce bioactive antibody-gold conjugate. This study built a base of colloidal gold immunochromatographic assay (GICA) kit for TCs residues.The o-tolidine and dicyclohexylcarbodiimide methods were applied to synthesize tetracyclines artificial antigen and coat antigen. Tetracycline, oxytetracyline, chlortetracycline and doxycycline were conjugated with carrier protein bovine serum albumin (BSA) and ovalbumnin (OVA) to synthesize artificial antigen and coat antigen respectively. The coupling ration was confirmed by UV spectrum, was employed to analyze the immunogens with supposed coupling ration.The artificial antigens were injected into Balb/c female mice and the blood samples were obtained from tails of Balb/c mice after injection and detected by iELISA method. When the titers of antiserum by iELISA were more than 1:6400, the fresh spleen cells of immunized mice were used for fusion with myeloma cells SP2/0. Positive cells were determined using iELISA. The positive cells were cloned and the subtype of monoclonal antibody was detected.Large amount of oxytetracycline and chlortetracycline monoclonal antibodies were obtained from ascites of BALB/C mice inoculated with the hybridomas. Caprylic acid-ammonium sulphate precipitation method was employed to purificate monoclonal antibody. Then they were coupled with colloidal gold to form bioactive oxytetracycline and chlortetracycline antibody-gold conjugate.