Study Of Enzyme Immunoassay For Detection Of Furazolidone Metabolite In Food

At present,the use of antibiotics in livestock and poultry breeding and aquaculture industry is still extensive and necessary.As a kind of banned veterinary drugs,nitrofurans can be used to treat animal gastrointestinal diseases,and now they are still used illegally in animal breeding industry.Nitrofurans can be quickly metabolized and presented chronically and steadily in the form of their metabolites after they are taken in animal bodies,and the metabolites will cause chronic poisoning,even have potential carcinogenic effects.Therefore,it is necessary and important for government supervision departments and breeding enterprises to detect the residues of nitrofurans metabolites.Furazolidone is a kind of nitrofurans,and its metabolite AOZ is used as a marker to determine the residues.The current methods for detection of furazolidone metabolite AOZ can be attributed to instrument detection methods and enzyme immunoassay.The former mainly includes high performance liquid chromatography(HPLC)and high performance liquid chromatography-tandem mass spectrometer(HPLC-MS/MS).Enzyme immunoassay includes enzyme linked immunosorbent assay(ELISA),chemiluminescent enzyme immunoassay(CLEIA)and biotin-avidin enzyme linked immunosorbent assay(BA-ELISA).There are a large amount of samples to be determined in daily food safety spot check tasks,which requires timely detection,instrument detection methods have difficulties in realizing above-mentioned demands since the slow detection speed and complex operations are needed.While enzyme immunoassay is simple and quick for detection,it has been widely used in the determination of drugs residues.This study established ELISA,BA-ELISA,and CLEIA methods for detection of furazolidone metabolite AOZ in animal derived food.Firstly,the hapten CPAOZ was synthesized by conjugating AOZ with 4-CBA.Then made CPAOZ conjugated with OVA by using N-hyduoxysuccinimide ester method to synthesize CPAOZ-OVA.Then CPAOZ was identified by infrared absorbance spectrum and nuclear magnetic resonance spectroscopy,CPAOZ-OVA was identified by UV spectrum.Secondly,the main conditions of three enzyme immunoassay methods were optimized.The optimized reaction conditions for ELISA were as follows: The dilution ratios of CPAOZ-OVA and monoclonal antibody were 800-fold and 6400-fold respectively;blocking solution was 1% skimmed milk;competitive reaction time was 60 min;incubation time for HRP-IgG was 45 min;chromogenic reaction time 15 min.The optimized reaction conditions for BA-ELISA were as follows: the dilution ratios of CPZOZ-OVA and monoclonal antibody were 2000-fold and 6400-fold respectively;the dilution ratio of SA-HRP was 8000-fold;blocking solution was 1% skimmed milk;competitive reaction time was 30 min;incubation time for Biotin-IgG was 60 min;incubation time for SA-HRP was 60 min;chromogenic reaction time was 15 min.The optimized chemiluminescence fluid's formula: Liquid A was 6 mmol/L of iodine phenol solution and 10 mmo/L luminol solution mixed by the same volume;Liquid B was 5mmol/L urea hydrogen peroxide solution;mix the same volume Liquid A and Liquid B before using.The optimized reaction conditions for CLEIA were as follows: the dilution ratios of CPAOZ-OVA and monoclonal antibody were 4000-fold and 1600-fold respectively;blocking solution was 1% skimmed milk;competitive reaction time was 30 min;incubation time for HRP-IgG was 60 min.Finally,the sensitivity,specificity,accuracy and precision of three enzyme immunoassay methods were evaluated.And the accuracies of these three methods were compared with national standard detection method HPLC-MS / MS.The linear equation for ELISA was Y=-0.253X+1.336(R2=0.995),IC50 was 2.015 ng/m L,the linearity range was 0.131~30.911 ng/m L,the recovery rate in negative pork samples was between 84.8%~90.3%.The intra-assay and inter-assay coefficients of variation were 3.3%~4.1% and 4.6%~6.7% respectively.The linear equation for BA-ELISA was Y=-0.267X+1.243(R2=0.999),IC50 was 0.606 ng/m L,the linearity range was 0.046-8.061 ng/m L.the recovery rate was between 88.4%~91.8%,the intra-assay and inter-assay coefficients of variation were 2.7%~4.6% and 4.8%~6.1%.The linear equation for CLEIA was Y=-0.357X+1.459(R2=0.984),IC50 was 0.486 ng/m L,the linearity range was 0.070~3.362 ng/m L,the recovery rate was between 89.3%~92.6%,the intra-assay and inter-assay coefficients of variation were 3.6%~4.8% and 3.2%~5.1%.The results showed that the sensitivities of BA-ELISA and CLEIA were comparable with that of national standard,while the IC50 of traditional ELISA method is relatively low.And the accuracies of three methods are close to that of national standard.Therefore,the enzyme immunoassay based on laboratory rapid quantitative detection can meet the rapid screening of AOZ in a large number of animal derived food.