Development And Preliminary Application Of Immunoassay Based On Gold Nanoparticles And Latex Beads For The Detection Of Kanamycin

Kanamycin is one of aminoglycoside antibiotics.Because of good bacteria growth inhibition,kanamycin has been widely used in medicine,agriculture,animal husbandry and aquaculture.Due to the abuse,the residual of kanamycin present in animal-derived food could eventually into the human body through the food chain.On the other hand,kanamycin could emission into water or soil in the form of prototypes or metabolites.Therefore,it is necessary to establish a high sensitivity,specificity,stability,simple and fast analysis method to detect kanamycin in food and the environment.In this study,the monoclonal antibodies with high specificity against kanamycin were successfully prepared.Direct competitive ELISA methods based on HRP and gold nanoparticles-HRP signal amplification for determination kanamycin were established.We also try to develop the new analysis methods based on latex beads.The immunogen Kana-BSA and coating antigen Kana-OVA were successfully synthesized by EDC method.The Balb/C mice were immunized subcutaneously with immunogen.The immunized mouse with the highest inhibition rate and good serum titer was selected to prepare hybridoma cells.After fused with SP2/0 cells,positive clones were screened by indirect competitive ELISA.Ten hybridoma cells were obtained.The immunoglobulin subtypes of the ten monoclonal antibodies were identified as IgG1 with Kappa light chain.The following methodology research was carried out using monoclonal antibody named A10E5.Kana-HRP was synthesized and a direct competitive ELISA method based on HRP was established.The the IC50 was 2.15 ng/m L,the LOD was 0.13 ng/m L and the linear range was 0.40~9.67 ng/mL.Au-HRP-Kana was also synthesized and a direct competitive ELISA was established based on Au-HRP as signal amplification molecule.The IC50 was 1.05 ng/m L,the LOD was 0.16 ng/m L and the linear range was 0.31~2.8 ng/mL.The cross reactivity of A10E5 monoclonal antibody was 99.07% with tobramycin and below 0.3% with other structural analogues.The rate of recovery in water,soil and milk was 88.6%~132.8%,and the coefficient of variation was less than 15.8%.The results showed that the content of kanamycin was 0.45~2.78 ng/mL in the water samples from Jiangsu University and 0.35~0.55 ng/mL in milk samples,while the content of kanamycin in the soil was not detected.The latex beads were conjugated with goat anti-mounse antibody using PEG as linker.The non-specific binding of latex beads was discussed and the experimental conditions were optimized.A quantification method based on latex beads was established by centrifugal.The IC50 was 4.66ng/m L,the LOD was 0.25 ng/mL,and the linear range was 0.73~19.07 ng/m L.Qualitative analysis of kanamycin was achieved by membrane filtration,the detection limit was 1 ng/mL obtained by naked eye.Finally,four methods for the detection of kanamycin were compared.It was found that the direct competitive ELISA method based on Au-HRP signal amplification was the most sensitive.It could be applied to detect for kanamycin in environmental samples.The qualitative method based on latex beads was simple and convenient without instruments,which could be applied to screen for the actual samples.