The Enzyme-Linked Immunosorbent Assay For The Detection Of Metronidazole

The direct competive ELISA and the direct competive enzyme-linked immunosorbent assay with enhanced chemiluminescent (CLEIA) detection of metronidazole and nitroimidazoles in animal derived food were developed.The first immunogen was obtained from metronidazole’s structural analogues:1-(2-aminoethyl)-methyl-5-nitroimidazole hydrochloride. Because the amino on the imidazole ring can be directly connected to the carrier protein by glutaraldehyde method. The other synthesises of haptens started from metronidazole itself. One is made by succinic anhydride method, we add succinic anhydride between metronidazole and carrier protein to make a7atomic chain length complete antigen. For the Linker method, we choosed the Thyroblobulin bovine protein as a carrier protein and make it connected to2,2-(ethylene two oxygen) dual (ethylamine), then connected metronidazole to N-N succinimide base carbonate, finally we continue to connect both of the compounds to form a extreme long chain antigen.10complete antigens from all these methods had been made.According to the different immune plan, we injected New Zealand big-eared white rabbit by differet complete antigen. The highest specificity and sensitivity of polyclonal antibody was obtained through non-chain link method. Using the method of mixed anhydride, we prepared the enzyme conjugate by linking MSA with horseradish peroxidase.Parameters of these two methods were optimized, including the concentration of coating-antibody, dilution of enzyme conjugate, blocking reagent, pH and Luminous substrate solution,Luminous enzyme label plate. Under the optimum conditions, the direct competive ELISA for metronidazole was developed. The sensitivity (IC50) and limit of method detection (IC15) were0.30±0.04μg/mL,0.036±0.003μg/mL, respectively. The metronidazole CLEIA was developed, the sensitivity (IC50) and limit of method detection (IC15) were0.022±0.04μg/mL,0.008±0.003μg/mL, respectively.The cross-reactivity against other nitroimidazoles is DMZ194%, TNZ76%, RNZ115%in direct competive ELISA. The recoveries of metronidazole from chicken muscle, shrimp were ranged from58.54%to111.64%in direct competive ELISA at three concentrations of1.2μg/g,6μg/g,20μg/g.The result indicates the direct competive ELISA and CLEIA method is high sensitivite, specific, it is highly suitable for the detection of metronidazole and nitroimidazoles residue in animal tissues.