Establishment Of A Highly Sensitive Magnetic Immunochromatography Assay For Amantadine Residues In Chicken Tissues And Eggs

Amantadine(AMD)is a synthetic drug for the treatment of human influenza and Parkinson’s disease,which has not been approved for use in livestock and poultry.According to the official announcement,AMD remains widely in chicken tissues and eggs.Long term exposure to AMD can damage human respiratory,reproductive and nervous systems.In addition,the abuse of AMD will enhance the resistance of influenza virus to it,increase the possibility of virus mutation,and reduce the effect of treatment.Therefore,it is necessary to monitor AMD residues in chicken tissues and eggs.The existing detection methods of AMD are mainly instrument detection and immunoassay.The pretreatment of instrument method is complex and not suitable for on-site detection.The immunoassay is simple,rapid and sensitive.Immunochromatography is the mainstream of rapid detection methods.In recent years,the combination of Magnetic nanoparticles(MNPs)and immunochromatography has become a research hotspot.The magnetic immunochromatographic strip has the characteristics of strong signal penetration,low biological background interference and higher sensitivity.Therefore,the establishment of magnetic immunochromatographic strip for AMD in chicken tissues and eggs will provide a more concise,efficient and sensitive detection method for the market,which has important application value.The main contents of this study include the following aspects:1.MNPs were synthesized by solvothermal method.The magnetic properties,morphology,size,surface potential and constituent elements were characterized by external magnetic field,scanning electron microscope,nanoparticle size /zeta analyzer and infrared spectrometer.The results showed that MNPs with uniform particle size,good dispersion and carboxyl groups on the surface could be synthesized in one step.2.Screening the coupling conditions between MNPs and monoclonal antibody of AMD to prepare MNPs-m Ab.The results showed that when the coupling solution was deionized water,the reconstitution solution was 0.5% BSA,the MNPs : m Ab was 100 μg : 2 μg,the coupling and blockling time were both 30 min,the MNPs-m Ab was stored by vacuum freezedrying method,the chromatography of the MNPs-m Ab was the most smooth on the strip.3.Optimize the factors affecting the sensitivity of MICA.The results showed that the sensitivity of the magnetic immunochromatography assay(MICA)was the highest when the nitrocellulose membrane was PALL VIV9025 100 R,the chromatographic solution was0.01 mol/L PBS(p H7.4),the secondary antibody(C-line)was 0.7 mg/m L,the antigen(Tline)was 0.05 mg/m L,the reaction time was 2 min,and the detection time was 7 min.4.The stability study showed that the strip and MNPs-m Ab microwells could be stored for 60 days at 25 ℃ or 30 days at 50 ℃,which sensitivity didn’t change.According to Arrhenius equation,the shelf life is 1 year at least.5.In the application of MICA,the qualitative limit of detection(LOD)for AMD in chicken and egg was 0.5 μg/kg.The accuracy of detection was 100% with 20 blank samples and 20 spiked samples of chicken and eggs from different sources.6.Add standard solution of AMD to chicken and egg blank samples according to1× LOD,2× LOD,4× LOD.The results showed that the coefficient of variation for T/C was less than 10% when detecting samples with the same concentration,which met the requirements of strip on rapid detection.7.In the comparative assessment of methods,white feather broilers were fed with AMD at 100 mg/kg,50 mg/kg and 25 mg/kg,and their liver and chicken were taken for detection.The results of MICA were consistent with LC-MS/MS,which indicated that the MICA established in this study can be used as a reliable tool for the detection of AMD residues in actual samples.In this study,the solvothermal method was used to synthesize MNPs and prepare MNPs-m Ab.After optimizing the conditions,a highly sensitive MICA for the rapid detection of AMD residues in chicken and eggs was established for the first time,which made up for the deficiency of the existing detection methods.It provides a reference for the establishment of MICA in the future,and has important academic significance and practical value.