Research On ELISA For Rapid Analysis Of Nitrofuran Metabolite Residues In Aquatic Products

Because of being cheap and easily obtained, Nitrofuran drugs, which belong to a class of synthetic antibiotics, are widely used in the disease prevention and treatment in aquaculture. Nitrofuran drugs can be rapidly metabolized in vivo, but their metabolites can steadily exist. The nitrofuran metabolite residues in aquatic products seriously threat to the health of consumers, so the nitrofurans are worldwide banned from use as fishery drugs.Now the analytical techniques for detecting nitrofuran metabolites by LC-MS/MS have been established. However, this method requires expensive equipment, complex pre-treatment, long analysis period and high cost, so it is not applicable for on-site analysis. The purpose of the research was to explore a new way to simultaneously detect several nitrofuran metabolites and to initially develope an ic-ELISA method for nitrofuran metabolites in aquatic products.First, the AOZ, a major metabolite of furazolidone, was synthesized by the condensation reaction of 2-hydroxyethyl hydrazine and dimethyl carbonate under the catalysis of sodium methoxide. Then three nitrofuran metabolites, AOZ, SEM and AHD, were derivated by carboxyl modification and they were sequentially coupled to HSA to obtain immunogen via the active ester method. And the 4-CPAOZ, 4-CPSEM and 4-CPAHD was coupled with OVA to get the coating antigens via the mixed anhydride method. The haptens, immunogens and coating antigens were identified through TLC, UV spectrum and LC-MS/MS spectrum. The coupling ratio was determined as 4:1,11:1 and 14:1 by UV spectrum scanning.Secondly, the synthesized immunogen(4-CPAOZ)4-(4-CPSEM)11-(4-CPAHD)14–HSA was immunized New Zealand white rabbits to get the antiserum simultaneously selective to 4-CPAOZ, 4-CPSEM and 4-CPAHD. After caprylic acid- ammonium sulfate precipitation and dialysis desalination, I got the purified McAb. And the antibody titers were 1:16000, 1:8000 and 1:10000 via checkerboard titration experiment and ic-ELISA.Thirdly, the ic-ELISA for nitrofuran metabolites were established by using the McAb. After checkerboard titration experiment and sequential parameter analysis, the several important parameters of ELISA test were optimized and the optimal reaction condition of the ELISA test was got. The regression equation of ic-ELISA for NPAOZ was y =-42.5595 x + 66.4298, R2 = 0.9866, and linear range was 0.5~10 ng/mL. The regression equation of ic-ELISA for NPSEM was y =-65.0732 x + 100.9356, R2 = 0.9842, and linear range was 2~25 ng/mL. The regression equation of ic-ELISA for NPAHD was y =-61.5168 x + 95.4171, R2 = 0.9879, and linear range was 2~25 ng/mL. The recovery of spiked blank samples of mandarin fish were between 80% an 120%, and the CVs were under 15%. It implied that the method was suitable for detection of nitrofuran metabolites in the aquatic products and it created good basis for the further development of commercial kits.