Modified 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)method was employed to synthesize the artificial antigen of norfloxacin(NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody(pAb).Ba...Modified 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)method was employed to synthesize the artificial antigen of norfloxacin(NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(icELISA) standard curve was established.This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml,with the half maximal inhibitory concentration(IC50)and limit of detection(LOD)values of 2.7 ng/ml and 0.06 ng/ml,respectively.The produced pAb exhibited high cross-reactivity to fluoroquinolones(FQs)tested,and the IC50 values to enoxacin,ciprofloxacin,and pefloxacin were 3.1,3.4,and 4.1 ng/ml,respectively.It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10%and 30%.When spiked in milk at 5,20,and 50 ng/ml,the recoveries for NOR,enoxacin,ciprofloxacin,and pefloxacin ranged 90.5%-98.0%,84.0%-95.2%,94.0%-106.0%,and 89.5%-100.0%,respectively.The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.展开更多
基金Project supported by the Henan Innovation Project for University Prominent Research Talents(No.2010HASTIT026)the Key Scientific & Technological Project of Education Department in Henan Province of China(No.2011A230003)
文摘Modified 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)method was employed to synthesize the artificial antigen of norfloxacin(NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(icELISA) standard curve was established.This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml,with the half maximal inhibitory concentration(IC50)and limit of detection(LOD)values of 2.7 ng/ml and 0.06 ng/ml,respectively.The produced pAb exhibited high cross-reactivity to fluoroquinolones(FQs)tested,and the IC50 values to enoxacin,ciprofloxacin,and pefloxacin were 3.1,3.4,and 4.1 ng/ml,respectively.It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10%and 30%.When spiked in milk at 5,20,and 50 ng/ml,the recoveries for NOR,enoxacin,ciprofloxacin,and pefloxacin ranged 90.5%-98.0%,84.0%-95.2%,94.0%-106.0%,and 89.5%-100.0%,respectively.The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.
