Establishment Of Rapid Immunological Detection Methods For Nitrofuran Metabolites

Although nitrofuran antibiotics were banned from use more than two decades at home andabroad for their potential teratogenic, carcinogenic and mutagenic effects, there are stillnitrofuran residues nowadays. This study aimed to development a fast detecting methodbasing on immunological principles, so great efforts were made to prepare monoclonalantibodies of nitrofuran metabolites with high sensitivity and specificity, then icELISA wasestablished、optimized and validated.To begin with, haptens were synthesized and analytes were derived from nitrofuranmetabolites with oxoacetic acid,4-carboxybenzaldehyde,4-nitrobenzaldehyde, and confirmedby Maldi Synapt QT of MS; Active ester method and mixed acid anhydride method wereemployed to obtain complete antigens by coupling haptens to proteins and characterized byUV.Mice were immunized with the antigens. The mice with high titer and good inhibitionafter several times of immunizations were selected, their spleens were token to undergo thecell fusion and the best cell lines were selected, ascites was produced and purified, in this waymonoclonal antibodies were obtained.IcELISA methods were established and coating antigens, analytes solvents, the pH andNaCl concentrations of analytes diluting solution were optimized and the sensitivity,specificity, accuracy of the optimized icELISA were detected. The results from optimizationshowed that the pH and NaCl affected a lot on the IC50of antibodies. The IC50of SEM, AHD,AOZ, AMOZ were successfully decreased from0.061ng ml-1,0.17ng ml-1,0.12ng ml-1,0.54ng ml-1to0.011ng ml-1,0.07ng ml-1,0.10ng ml-1,0.54ng ml-1by optimization. And theLOD were0.0013ng ml-1,0.022ng ml-1,0.011ng ml-1,0.067ng ml-1, respectively.