The Research On The Rapid Detection Assay Of The Residues Of Four Nitrofuran Drugs

Nitrofurans were widely used in livestock diseases’ prevention and treatment, or as additives to promote animal growth. Beacause of nitrofurans have carcinogenic and mutagenic potential dangerous for humans, which had been forbidden in animal breeding. But it is still used by illegal because of its wide antibacterial and low price, especially furazolidone, furaltadone, nitrofurazone and nitrofurantoin. At present most of the reported immunological detection methods are enzyme-linked immunosorbent assay(ELISA). While there are a few reports about the direct competitive chemiluminescent enzyme immunoassay(dc-CLEIA). Most reports of colloidal gold immunochromatographic assays are single antibiotic analyte detection, while can simultaneously detect multiple residues has become research trend. The purpose of this paper is to establish SEM, AMOZ, AHD and AOZ direct competitive chemiluminescence enzyme immunoassay, and simultaneously to detect SEM, AHD, AMOZ and AOZ colloidal gold immunochromatographic method.The dc-CLEIA method for SEM, AMOZ, AHD and AOZ. CPAOZ-HRP、CPAMOZ-HRP、CPAHD-HRP and CPSEM-HRP were synthesized by carbodimiide method. According to the AMOZ dc-CLEIA method: with a linear working range between 0.0625 and 8 ng/mL, the 50% inhibitory concentration(IC50) was 0.289 ng/mL, the regression equation was y =-0.397 x + 0.286,(R2 = 0.992); the coefficient variation of intra and interbatch were 2.24% and 3.21%, respectively; the minimum detection limit of NPAMOZ in the samples was 0.0596 μg/kg, the rates of recovery of sample addition were 85%-91.93%. According to the AOZ dc-CLEIA method: with a linear working range between 0.0625 and 8 ng/mL, the 50% inhibitory concentration(IC50) was 0.206 ng/mL, the regression equation was y =-0.329 x + 0.274(R2 = 0.992); the coefficient variation of intra and interbatch were 3.31% and 5.48%, respectively; the minimum detection limit of NPAOZ in the samples was 0.1052 μg/kg, the rates of recovery of sample addition were 78%-103%. According to the SEM dc-CLEIA method: with a linear working range between 0.0625 and 8 ng/mL, the 50% inhibitory concentration(IC50) was 0.271 ng/mL, the regression equation was y =-0.35 x + 0.302(R2 = 0.991); the coefficient variation of intra and interbatch were 2.37% and 2.6%, respectively; the minimum detection limit of NPSEM in the samples was 0.1165 μg/kg, the rates of recovery of sample addition were 82%-95%. According to the AHD dc-CLEIA method: with a linear working range between 0.125 and 32 ng/mL, the 50% inhibitory concentration(IC50) was 0.625 ng/mL, the regression equation was y =-0.358 x + 0.427(R2 = 0.992); the coefficient variation of intra and interbatch were 3.86% and 5.93%, respectively; the minimum detection limit of NPAHD in the samples was 0.3635 μg/kg, the rates of recovery of sample addition were 75%-94%.Colloidal gold immunochromatographic assay for simultaneously detecting SEM, AHD, AMOZ and AOZ. The detection limit of SEM, AHD, AMOZ and AOZ were 1, 1, 1 and 0.75 ng/mL, respectively. There were no cross-reactivity with other nitrofuran parents and its metabolites, sulfa, metronidazole, et al. The rate of false positive in 20 blank samples was 0 by colloidal gold strip, the rate of false negative in 50 positive samples was 0 by colloidal gold strip. The colloidal gold strip test results of the actual sample was consistent with the results of ELISA, which indicated that the method could be used for detecting SEM, AHD, AMOZ and AOZ.