Development Of Several Novel Immunoassay Methods For Aminoglycoside Antibiotics And Preliminary Application

Aminoglycoside antibiotics（AGs）are widely used in medicine,animal husbandry and aquaculture due to their good antibacterial activity.These used antibiotics may enter the environment through wastewater or waste garbage,which would endanger ecological balance and human health through environmental media and food chain migration.Therefore,it is particularly essential to explore high-throughput,rapid,simple and sensitive methods for the detection of AGs in the environment.In this study,using kanamycin（KAN）and streptomycin（STR）,two typical AGs,as research objects,highly specific monoclonal antibody against streptomycin was prepared and ELISA for the detection of STR was established.Following then,several novel immunoassay methods combined with new nanomaterials were developed for the detection of KAN and STR in the environment,where monoclonal antibody against KAN had been prepared in our laboratory.And these novel immunoassay methods were applied for the analysis of environmental samples.Immunogen（STR-BSA）and coating antigen（STR-OVA）were synthesized by EDC method,and 5 strains of hybridoma cells capable of secreting monoclonal antibody against STR were successfully screened by hybridoma technology.The hybridoma cell,6H3D5,showed the best inhibitory effect and no cross-reactivities with other AGs except dihydrostreptomycin.After optimizing the conditions,the standard curve of STR was established and the inhibition of concentration(IC₅₀),limit of detection（LOD）and detection range(IC₂₀^IC₈₀)of the standard curve were 2.72ng/mL,0.183 ng/mL and 0.446¹6.5 ng/mL,respectively.In order to realize the simultaneous detection of multiple analytes,the Dual Dot-ELISA method was established in this study using the nitrocellulose membrane as the carrier for detecting KAN and STR.Under optimal experimental conditions,LOD of Dual-Dot ELISA was 0.9 ng/mL（KAN）and 6.25 ng/mL（STR）.After further quantization by ImageJ software,LOD for KAN and STR was 0.09 and 1.37 ng/mL.The Dual-Dot ELISA and UPLC-MS/MS results were also well correlated.This method was successfully applied to the detection of environmental water samples in Jiangsu university.The results showed that the concentration of STR in all samples was lower than LOD of Dual Dot-ELISA,and there are four water samples containing KAN,ranging from 0.52¹.47 ng/mL.Based on gold nanoparticles and carbon nanotubes assisted multi-enzyme particles,magnetic bead immunoassay methods for detecting KAN were developed.AuNPs and MWCNTs were used as carriers to synthesize Au/KAN-HRP/HRP,MWCNTs/KAN-HRP/HRP-p and MWCNTs/KAN-HRP/HRP-c,and then the catalytic efficiency of multi-enzyme probes was compared,MWCNTs/KAN-HRP /HRP-p>MWCNTs/KAN-HRP/HRP-c>Au/KAN-HRP/HRP.Three high-sensitivity immunoassay methods were established based on three kinds of multi-enzyme probes（Au/KAN-HRP/HRP,MWCNTs/KAN-HRP/HRP-c and MWCNTs/KAN-HRP/HRP-p）,and the sensitivity was 2,15 and 37 folds higher than the traditional IMBs method,respectively.The results showed that the concentration of KAN in the environmental water sample was 0.935¹.055 ng/mL,and in the milk sample was 0.096⁰.132ng/mL,while no KAN was detected in soil samples.In order to realize the rapid detection of streptomycin residues in the environment,an enhanced immunochromatography sensor based on Au@Pt nano-enzyme was established.Au@Pt nanomaterials with enzyme-like activity were synthesizedbyone-stepmethodandcharacterizedbyTEM.An immunochromatographic sensor based on Au@Pt was established by coupling monoclonal antibody against STR on Au@Pt nanozyme.After optimization,the LOD was 1.0 ng/mL by naked eye interpretation and 0.9 ng/mL for quantization by ImageJ software.An enhanced immunochromatographic sensor based on Au@Pt nanozyme was constructed by the aid of enzyme-like activity of Au@Pt nanozyme and using AEC as substrate.After optimization,the LOD of naked eye interpretation was 0.1ng/mL,and after quantification,the LOD was 0.06 ng/mL,which was about 80 times more sensitive than traditional colloidal gold chromatography.