Preparation And Application Of Lomefloxacin Immunochromatographic Test Strip Based On Gold Nanoflowers

Lomefloxacin(LOM)is a synthetic third-generation quinolone antibacterial.It is commonly used to treat bacterial and mycoplasma infections,due to its broad-spectrum and highly effective bactericidal effect.The abuse of LOM will lead to the increased resistance of bacteria.However,the residual LOM in food can enter the human body through the food chain and endanger human health.In announcedmentt No.2292 issued in 2015,China's ministry of agriculture has explicitly prohibited LOM and other related preparations from being used in animal food.Therefore,it is particularly important to establish a simple and rapid method to detect the residual LOM in food.Immunochromatographic test strips(ICTS)are widely used in medical testing and food safety rapid testing,because they have the advantages of simple operation,rapid detection,environmental protection,safe and strong specificity.Among the markers of ICTS,colloidal gold is still one of the most widely used markers.The gold nanoflowers(AuNFs)has a strong localized surface plasma response due to its unique spike structure.Meanwhile,AuNFs can bind more antibodies because of its larger specific surface area,which can effectively improve the detection sensitivity.In this study,AuNFs was used as a detection marker.After systematic research,a highly sensitive AuNFs immunochromatographic assay(ICA)was established to detect LOM in milk rapidly and quantitatively.At the same time,an industrial plant of the LOM AuNFs-ICTS was designed.Firstly,this study planned to prepare anti-LOM monoclonal antibodies(mAb).LOM hydrochloride was used as the raw material,the complete antigens of LOM-BSA and LOM-OVA were prepared by the EDC/NHS carbodiimide method.To evaluate the effect of antigen preparation,the immunogen LOM-BSA and the coated original LOM-OVA were characterized by the ultraviolet spectrophotometer.The mice for experimental were immunized with LOM-BSA,and the anti-serum titers of the immunized mice were detected by enzyme-linked immunosorbent assay.The experiment was monitored from the three times immunization of mice.Shock immunization selection was performed after the fifth immunization of mice,followed by cell fusion.After continuous operations such as cultivation,screening and subcloning,the anti-LOM mAb may be obtained.Secondly,an AuNFs ICA for quantitative detection of LOM was established.The results showed that the optimal labeling pH of LOM antibody was 6,the optimal antibody labeling concentration was 4 ?g/mL,and the detection time was 13 min.To detect LOM in PBS solution,the linear regression equation was:y=-0.321g(x)+ 0.255(R2=0.9848),linear range was 0.4-4 ng/mL,IC50 was 0.1715 ng/mL,LOD was 0.0117 ng/mL.The standard curve for detecting LOM in milk was y=-0.4761g(x)+0.2644(R2=0.9861),the linear range was 0.2-2 ng/mL,the IC50 was 0.3199 ng/mL,and the LOD was 0.0528 ng/mL.In this method,the recovery rate within the batch was 94.2%-105.3%,and the coefficient of variation was 2.69%-10.60%;the recovery rate between batches was 96.7%-105.0%,and the coefficient of variation was 2.26%-6.90%.The established method achieves a highly sensitive detection of LOM,which was of great significance for effectively monitoring the use of LOM in food.Finally,in order to realize the industrialization of the established AuNFs LOM ICA,a production plant for the LOM ICST was designed.And its products can be applied to the food safety rapid detect industry,thereby ensuring food quality and safety.While,the plant was designed with the principles of reasonableness,economy,efficiency,and compliance with national and industry standards.The design content included plant site selection,process part,building part design,public system,safety,healthy,environmental protectionand so on.And it provides a process flow chart and factory general plan layout.In this paper,the researches started with the preparation of experimental materials to prepare anti-LOM mAb and AuNFs ICTS,finally realized the industrialization of the ICTS.It was expected that it can be applied to the actual detection of agricultural products or food to ensure the food safety.