Preparation Of Magnetic Immunochromatographic Test Strip And The Application In Rapid Detection Of Fluoroquinolones

Fluoroquinolones are commonly used antibiotics in veterinary clinic,but excessive drug residues in animal food will endanger food safety and threaten the health of consumers and the safety of ecological environment.It is important to establish a sensitive and rapid detection method to ensure quality of animal derived food.In this study,magnetic nanoparticles（MNPs）were used to label monoclonal antibodies,and the lateral flow immunoassay（LFIA）test strips were prepared.A rapid method for the determination of fluoroquinolones（FQs）in milk was established.The MNPs labeled antibody was prepared by carbodiimide method and characterized by FTIR.The coupling rate of monoclonal antibody was determined by BCA method and the biological activity of MNPs labeled antibody was analyzed.FTIR spectra showed that the coupling products had absorption peaks at 1391 cm^-1and 1651 cm^-1,indicating the existence of amide bond.By measuring the adsorption capacity of the labeled antibody,it was further verified that the magnetic microspheres were successfully coupled with the antibody,and the MNPs labeled antibody still maintained biological activity,which could recognize and bind the tested substance in a short time.The factors such as the amount of antibody and reaction time were optimized.It was found that when the amount of antibody was 40μg,the coupling rate was higher and the reaction between antibody and MNPs was more sufficient.When the reaction time was controlled between 2-4 h,the activity of MNPs labeled antibody was not destroyed.Using MNPs labeled antibody as recognition element,a rapid and reliable LFIA test strip was prepared.The composition of the test strip,the coating concentration of antigen and goat anti-mouse Ig G,as well as the components in the pretreatment buffer were optimized.By comparing different types of NC films,it was found that Vivid 90 NC membrane has better color rendering effect,moderate line width and higher sensitivity.By exploring the coating concentration of test line（T line）and control line（C line）,it was found that the color of T line was the best at the concentration of 0.8 mg/m L antigen and 0.8 mg/m L goat anti-mouse Ig G.The color intensity of C line and T line was the same in this case,indicating higher sensitivity of the method.After that,several factors such as surfactant,sugar molecule,polymer and Na Cl were optimized.It was determined that the buffer containing 4%Tween-20,10%sucrose-5%trehalose and 0.1%PVP was the best pretreatment system for the sample pad and conjugate pad.No positive effect of Na Cl on LFIA was observed,consequently,no Na Cl was added.The residual FQs drugs in milk were analyzed by MNPs LFIA test strip.The linear range of 10 FQs ranged from 0.90477 to 0.9992,and LODs were from 0.063 to 0.756 ng/m L.The sensitivity of the method was satisfactory,which could meet the requirements of the detection.The recoveries of difloxacin and obifloxacin were low.The recoveries of the other eight FQs ranged from 31.42%to 83.67%,with RSD ranging from 0.12%to 7.13%.The results of milk samples and raw milk samples were negative,and no FQs drug residues were detected.The analysis results were consistent with those of UPLC-MS/MS.