Study On The Immunoassay Technolgies Of Quinolone Antibiotics In Water

In recent years,antibiotics have been frequently detected in water samples around the world because of the abuse of antibiotics.It does harm to human health and ecological environment.A rapid,accurate and robust detection technology for antibiotics was needed to evaluate the level of antibiotics in the environment and to prevent the pollution of antibiotics.This study aims to establish two types of immunoassay technology including immunosorbent assay method and planar waveguide immunosensor to detect antibiotics,which were based on specific interactions between antigens and its antibodies.Firstly,a carbodiimide method was developed to synthesize the enrofloxacin complete antigen.The complete antigen posseses good performance,it has a concentration of 1.23 mg/mL and a conjugation ratio between 14 and 16.Using the complete antigen,four IgG/?types of enrofloxacin monoclonal strains were successfully obtained by immunizing mice,two strains of them were choose for ascites preparation and antibody purification,and two monoclonal antibodies were successfully obtained.The antibody affinity constants were 4.56×10⁹ L/moL and 2.22×10¹⁰ L/moL respectively.Secondly,the immunoreaction kinetics were analyzed,and it was concluded that appropriate reduction of antibody concentration could improve the method's sensitivity.The suitable concentration of antigens and antibodies for four quinolone antibiotics were confirmed by chessboard assay,and the indirect competitive ELISA methods of four quinolone antibiotics were successfully established.The detection limit of enrofloxacin was 1.92?g/L using our independent developed antigen and antibody through indirect competitive ELISA method.Using the commercial antigen and antibody,the detection limit of enrofloxacin,norfloxacin,ciprofloxain and ofloxacin was 0.95?g/L,5.59?g/L,0.66?g/L and 13.31?g/L respectively.Thirdly,Planar waveguide immunosensor for detection of quinolone antibiotics was developed.It combined immunoassay technology and the sensor technology to detect antibiotics.The immunochip modification method and antibody fluorescent labeling method were explored.The parameters influencing the detection process have been optimized including the concentration of antibody,kinds of desorbent,pH and hardness.The single detection and simultaneous detection methods of enrofloxacin and norfloxacin were established,and the sensitivity of planar waveguide immunosesor was superior to immunosorbent assay method.For three kinds of real water samples with spiked enrofloxacin,the method exhibited good recoveries with 100±20%.Finally,the standardized protocol of enzyme-linked immunoassay?ELISA?for the detection of enrofloxacin was studied.The standardized program including application scope,method principle,reagent and material,instrument and equipment,sample preparation,analysis procedure,result calculation and presentation,quality assurance and quality control?QA/QC?and precautions were proposed,it provided support for the application of ELISA method to detect antibiotics in environmental monitoring.