| Enramycin,also called enduracidin,is a group of polypeptide antibiotics been utilized as antibiotic growth promoters(AGP)to add to poultry feeds to improve production in recent years.Although the advantages of enramycin in promoting animal growth,the extensive use may accumulate in animal products,resulting in antibiotic residues in animal derived food,causing the emergence of multidrug-resistant(MDR)bacteria and thereby threat human health.China have only set the concentrations of enramycin added in feed addictives,in Japan and korea,the maximum residue limit(MRL)for chicken muscle,liver and kidney was 30μg/kg.At present,the detection technology of harmful substances residues in food samples were mainly instrumental methods and immunoassay methods.Instrumental methods such as high performance liquid chromatography(HPLC)was time consuming and requiring expensive equipment and operations.Immunoassay mainly includes enzyme-linked immunoassay(ELISA)and colloidal gold method,having the advantage of high-throughput and less detection time.To the best of our knowledge,no papers have been reported to develop ic-ELISA methods for the analysis of enramycin in edible animal tissues.Thus,our study aimed to produce a monoclonal antibody(mAb)with high-sensitivity against enramycin and to establish an accurate ic-ELISA method based on monoclonal antibody to detect enramycin in edible animal products with a simple sample preparation procedure.The main research results are as follows:(1)Synthesis and identification of artificial antigenWe first analyze the two components from the angle antibacterial ability,then coupled the more biologically active Er-A to the carrier protein(BSA/OVA)via the glutaraldehyde(GA)and the carbonyldiimidazole(CDI)method at two different positions.UV spectrophotometry,FTIR and SDS-PAGE was used to confirm the successful conjugating.(2)Production and Characterization of the monoclonal antibody against Er-AThe two immunogens were used to elicit specific antibodies against the enramycin and a specific monoclonal antibody was achieved.Then concentrations of the heterologous coating antigen(7 μg/mL),the antibody dilution(1:8000)and the IgG-HRP dilution(1:5000)were determined by checkerboard method.The mAb(2H1F91)we got showed high titer of 32000 and the ICso value was 108.5 ng/mL.The antibody 2H1F91 showed CR towards Er-A(100%),Er-B(91.4%),and less than 0.01%with the rest analogues.(3)Optimization and development of ic-ELISAWe optimized factors of coating antigen concentration,pH of standard solution,concentration of methanol,Ionic strength of standard solution,reaction time of antibody and color development respectively.A method for detecting Enramycin was established.The detection limit was 12.056 ng/mL,working range was between 27.1-434 ng/mL,the spiked experiment indicated that the limits of detection was 144.8 μg/kg,98 μg/kg in swine and chicken matrix and the average recoveries of Er-A and Enramycin were 68.94%-122.86%and 72.32%-106.91%from spiked pork and chicken muscles,respectively.The coefficients of variation(CV)were less than 12%.The good correlation(r=0.9644)between HPLC and ic-ELISA results indicated this rapid and reliable ic-ELISA assay can be used to detect enramycin in pork and chicken muscle samples. |
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