Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb)....Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(ELISA) standard curve was established.This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg(R2=0.9567),with the half maximal inhibitory concentration(IC50) and limit of detection(LOD) values of 9.4 μg/kg and 0.2 μg/kg,respectively.Of all the competitive analogues,the produced pAb exhibited a high cross-reactivity to ciprofloxacin(CIP)(87%),the main metabolite of ENR in tissues.After optimization,the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork.The overall recoveries and coefficients of variation(CVs) were in the ranges of 86%-109% and 6.8%-13.1%,respectively.It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.展开更多
Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb)....Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(ELISA) standard curve was established.This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg(R2 =0.9567),with the half maximal inhibitory concentration(IC50) and limit of detection(LOD) values of 9.4 μg/kg and 0.2 μg/kg,respectively.Of all the competitive analogues,the produced pAb exhibited a high cross-reactivity to ciprofloxacin(CIP)(87%),the main metabolite of ENR in tissues.After optimization,the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork.The overall recoveries and coefficients of variation(CVs) were in the ranges of 86%-109% and 6.8%-13.1%,respectively.It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.展开更多
A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry （GC-MS） determination of 19-nortestosterone （19-NT） in animal tissues has been developed. In our optimized procedures, enzymat...A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry （GC-MS） determination of 19-nortestosterone （19-NT） in animal tissues has been developed. In our optimized procedures, enzymatic hydrolysis with β-glucuronidase from Escherichia coliwas performed in an acetate buffer （pH 5.2, 0.2 mol/L） Next, the homogenate was mixed with methanol and heated at 60℃ for 15 min, then placed in an ice-bath at -18℃ for 2 h. After liquid-liquid extraction with n-hexane, the analytes were subjected to a normal-phase solid phase ex- traction （SPE） C18 cartridge for clean-up. The dded organic extracts were derivatized with heptafluorobutydc anhydride （HFBA）, and then the products were injected into GC-MS. Using electron impact mass spectrometry （El-MS） with positive chemical ionization （PCI）, four diagnostic ions （mlz 666, 453, 318, and 306） were determined. A standard calibration curve over the concentration range of 1-20 ng/g was reached, with Y=467084X-68354 （R^2=0.9997） for 19-NT, and the detection limit was 0.3 ng. When applied to spiked samples collected from bovine and ovine, the recoveries ranged from 63% to 101% with relative standard deviation （RSD） between 2.7% and 8.9%. The procedure is a highly efficient, sensitive, and more economical method which offers considerable potential to resolve cases of suspected nandrolone doping in husbandry animals.展开更多
基金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-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(ELISA) standard curve was established.This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg(R2=0.9567),with the half maximal inhibitory concentration(IC50) and limit of detection(LOD) values of 9.4 μg/kg and 0.2 μg/kg,respectively.Of all the competitive analogues,the produced pAb exhibited a high cross-reactivity to ciprofloxacin(CIP)(87%),the main metabolite of ENR in tissues.After optimization,the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork.The overall recoveries and coefficients of variation(CVs) were in the ranges of 86%-109% and 6.8%-13.1%,respectively.It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.
文摘Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(ELISA) standard curve was established.This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg(R2 =0.9567),with the half maximal inhibitory concentration(IC50) and limit of detection(LOD) values of 9.4 μg/kg and 0.2 μg/kg,respectively.Of all the competitive analogues,the produced pAb exhibited a high cross-reactivity to ciprofloxacin(CIP)(87%),the main metabolite of ENR in tissues.After optimization,the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork.The overall recoveries and coefficients of variation(CVs) were in the ranges of 86%-109% and 6.8%-13.1%,respectively.It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.
基金Project supported by the Eleventh Five-Year Plan for National Science and Technology of China (No.2006BAK02A21/1)the Henan Innovation Project for University Prominent Research Talents (No.2010HASTIT026),China
文摘A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry （GC-MS） determination of 19-nortestosterone （19-NT） in animal tissues has been developed. In our optimized procedures, enzymatic hydrolysis with β-glucuronidase from Escherichia coliwas performed in an acetate buffer （pH 5.2, 0.2 mol/L） Next, the homogenate was mixed with methanol and heated at 60℃ for 15 min, then placed in an ice-bath at -18℃ for 2 h. After liquid-liquid extraction with n-hexane, the analytes were subjected to a normal-phase solid phase ex- traction （SPE） C18 cartridge for clean-up. The dded organic extracts were derivatized with heptafluorobutydc anhydride （HFBA）, and then the products were injected into GC-MS. Using electron impact mass spectrometry （El-MS） with positive chemical ionization （PCI）, four diagnostic ions （mlz 666, 453, 318, and 306） were determined. A standard calibration curve over the concentration range of 1-20 ng/g was reached, with Y=467084X-68354 （R^2=0.9997） for 19-NT, and the detection limit was 0.3 ng. When applied to spiked samples collected from bovine and ovine, the recoveries ranged from 63% to 101% with relative standard deviation （RSD） between 2.7% and 8.9%. The procedure is a highly efficient, sensitive, and more economical method which offers considerable potential to resolve cases of suspected nandrolone doping in husbandry animals.
