Preparation And Application Of The Generic Monoclonal Antibody For Doxycycline

Tetracycline drugs are a class of antibiotics that are usually used as veterinary drugs.For example, tetracycline, chlortetracycline, oxytetracycline and doxycycline are thecommonly used representatives for the treatment of various diseases in livestock andpoultry. As these drugs are widely used, their residues in the foods of animal originbecome a public concern. Among the detection methods for tetracyclines residues,immunoassay is widely used due to its merits of high specificity, simple, sensitive andfast.In this study, doxycycline (DC) and chlortetracycline (CTC) were derivatized with4-aminophenylacetic acid by the diazonium coupling reaction respectively to synthesizetwo new haptens (DC-C and CTC-C). The two haptens were coupled to bovine serumalbumin and ovalbumin respectively to prepare the immunogens (DC-C-BSA) and thecoating antigens (DC-C-OA and CTC-C-OA) by using the mixed anhydride method. Inthis coupling method, the introduced carboxyl group was used to link the hapten and thecarrier. The coupling ratio in immunogen DC-C-BSA was19and was15in coatingantigen DC-C-OA. The coupling ratio in coating antigen CTC-C-OA was9. Furthermore,doxycycline was coupled with bovine serum albumin and ovalbumin respectively toprepare the immunogen (DC-BSA) and the coating antigen (DC-OA) with thehomobifunctional agent1,4-butanediol diglycidyl ether as the linker, and the couplingratio in immunogen DC-BSA was16and was11in coating antigen DC-OA.The two immunogens were used to immunize8-week-old female Balb/c mice toproduce the monoclonal antibodies. Finally, two hybridoma cell strains excreting thespecific monoclonal antibody for doxycycline were obtained. The three coating antigens(DC-OA, DC-C-OA and CTC-C-OA) and the two antibodies (R3M1and R8M4) werearranged into six combinations (homologous and heterologous) to optimize the reagentscombination. After comparison of the performances of these combinations, aheterologous indirect competitive ELISA (antibody from immunogen DC-C-BSA andcoating antigens CTC-C-OA) was then used to determine the seven tetracyclines inbovine muscle and milk. The optimal concentration of coating antigen (CTC-C-OA) was 2.1μg/ml, and the optimal working dilution of the McAb was1:10000. Thecrossreactivities to the seven analytes were in the range of47%~102%and the limits ofdetection were in the range of1.5~6.9ng/mL depending on the compound. Therecoveries of the seven drugs from fortified blank samples were in the range of75.3%~106.8%with coefficients of variation lower than10.9%.Therefore, this method could be used as a multi-analytes screen tool for routinemonitoring the residues of these tetracycline drugs in bovine muscle and milk.