Study Of Enzyme Immunoassay For Furaltadone Metabolite

Veterinary drugs,which can promote growth,improve product quality,improve feed utilization,reduce the morbidity and mortality of animals,etc,is indispensable to modern animal husbandry.But in practical production,misuse,abuse of veterinary drugs lead to veterinary drug residues exceeded in animal products which were easy to accumulate toxicity,resulting in drug resistance and threaten to human health after enter human body through the food chain.Furaltadone is a kind of nitrofurans antibiotics,which will be rapidly metabolized to 3-amino-5-methylmorpholino-2-oxazolidinone in the body and can exist for a long time.The instrumental analysis have high sensitivity and low detection limits,and the equipment is expensive and the operation is complex which need professional personnel and time-consuming.In this study,we established enzyme linked immunosorbent assay(ELISA),biotin-avidin enzyme-linked immunosirbent assay(BA-ELISA)and chemiluminescence enzyme immunoassay(CLEIA)to detect AMOZ.The methods are enzyme immunoassay based on antigen-antibody specific responses.Enzyme immunoassay can meet the actual detection in terms of sensitivity,specificity,precision,accuracy and can be used for rapid screening of the sample with the characteristics of fast,simple,low cost.First,CPAMOZ was synthesized by derivatization of AMOZ with4-carboxybenzaldehyde(4-CBA),and identified by nuclear magnetic resonance and infrared spectroscopy.Artificial antigen CPAMOZ-OVA were obtained by CPAMOZ and ovalbumin(OVA)used the activate ester method and identified by UV scanning.Second,the main factors affecting the enzyme immunoassay were optimized.The optimal reaction conditions of ELISA were as follows:dilution of CPAMOZ-OVA and monoclonal antibody were 500-fold and2000-foold,respectively,the blocking solution was 2% skimmed milk,the competition reaction time and horseradish peroxidase labelled goat antimouselgG conjugate(HRP-IgG)incubating time were both 30 min,the chromogenic reaction time was 15 min.The chemiluminescence fluid of CLEIA method :iodine phenol solution and lumino with density of 6 mmol/L and 10 mmol/L,respectively,mixed by equal volume ratioi was A liquid;adding 4 μL 30%H2O2 to 10 mL Tris-HCl was B liquid.A and B liquid mixed equal volume before use.The optimal reaction conditions of CLEIA were as follows:dilution of CPAMOZ-OVA and monoclonal antibody were both 4000-fold,the blocking solution was 2% skimmed milk,the competition reaction time and HRP-IgG incubation time were both 30 min.The optimal reaction conditions of BA-ELISA were as follows: dilution of CPAMOZ-OVA and monoclonal antibody were 1000-fold and 2000-fold,respectively,streptavidin-horseradish peroxidase(SA-HRP)dilution was 8000-fold,the blocking solution was 2% skimmed milk,the competition reaction time and SA-HRP incubating time were both 30 min,Biotin-IgG incubated 60 min,and the chromogenic reaction time was 15 min.Finally,three methods of enzyme immunoassay were used to evaluate the sensitivity,specificity,precision and accuracy.The linear equation of ELISA was y=-0.439x+0.670,IC50 was 2.439 ng/mL,the linear range was0.506~11.765 ng/mL;the intra-assay and inter-assay coefficient of variation(CV)were 1.4%~5.8% and 2.1%~4.9%,respectively.The recovery was86.7%~ 90.1% and coefficient of variation was 4.8%~7.3%.The linear equation of CLEIA was y=-0.493x+0.471,IC50 was 0.873 ng/mL,the linear range was 0.215~3.545 ng/mL;the intra-assay and inter-assay CV were1.5%~4.3% and 1.2%~5.2%,respectively.The recovery was 88.9%~94.2%and CV was 4.2%~6.3%.The linear equation of BA-ELISA was y=-0.442x+0.483,IC50 was 0.915 ng/mL,the linear range was 0.192~4.367ng/mL;the the intra-assay and inter-assay CV were 1.3%~4.2% and1.1%~5.6%,respectively.The recovery was 92.5%~94.6% and CV was4.4%~9.6%.The recovery and CV of High-Performance liquid chromatography with tandem mass spectrometric(HPLC-MS/MS)were 98.7%~99.8%and 1.1%~2.6%,respectively.The cross-reactivity of the three enzyme immunoassays were lower than 0.1% with other structural analogues and derivatization.The three kinds of enzyme immunoassay for detection of AMOZ varied in terms of the sensitivity,precision and accuracy,the accuracy was slightly inferior to the HPLC-MS/MS,but can meet the actual need for sample testing and can be used for rapid screening of AMOZ in food.