Establishment And Application Of Colloidal Gold Immune Immunochromatography Detection For Enrofloxacin In Aquatic Products

Veterinary drug residues in aquatic products have become increasingly prominent,veterinary bring a lot of action in the breeding process, but have a great harm to humanbody after ingestion, so strictly control of veterinary drug residues in aquatic products isparticularly important. Enrofloxacin (Enrofloxacin, ENR) is a third generation quinolone,reaching maximum concentration fast in the tissue fluid, widely tissue distribution, largenumber and variety of antibacterial and many other advantages, and its metabolites alsohave a good antibacterial effect, it is widely used in aquaculture disease prevention. As partof the farmers there were irregularities medication, easily lead to the enrofloxacin go withinthe food chain into the human body, causing damage to the cartilage tissue, central nervoussystem, digestive system, etc., while larger doses can also cause sluggish nervous system,liver damage and skin allergies and other problem.Through the use of colloidal gold immunoassay method, to establish a on-site rapiddetection method for monitoring the ENR residuals in aquatic products. GICA method hasmany advantages, such as fast detection speed, accurate test results, etc., it’s very suitablefor on-site rapid detection.The study obtained the following results:1. ENR-OVA artificial antigen synthesis. Using N-hydroxysuccinimide active estermethod, ENR coupled to OVA. The conjugants were identified by FPLC, UV scanning andSDS-PAGE., shows that ENR is successfully coupled with OVA.2. Preparation of polyclonal antibodies. New Zealand rabbits were immuned withENR-OVA, obtained the antisera and purified by ammonium sulfate saturation measured.Estimated by indirect competitive ELISA, the titers were between1:8000and1:64000.The1st rabbit antiserum, which had higher titer, is used to established the indirectcompetitive ELISA method, when ENR concentration between0.1~8.1ng/mL, bindingrate B/B0as the vertical axis, and standard concentration as the horizontal axis，obtainedthe equation by fitting curve: y=-40.613lg(x)+58.963，R2=0.9959，IC50was1.662.3. Preparation of colloidal gold. Prepared20nm colloidal gold particles using theFrens method, adjusted pH to8.2with K2CO3, added2mL0.1mg/mL solution ofENR-PcAb drop by drop, then blocked with BSA, purified using high-speed centrifugation,finally obtained magenta colloidal gold solution.4. Preparation of the test strip. Colloidal gold labeled enrofloxacin polyclonal antibodysprayed65μL/cm2on the film; Millipore’s M180NC membrane; test line and control line (designated amount of the film were1.0μL/cm); ENR-OVA and goat anti-rabbit IgGconcentrations were coated with1000mg/L; treatment before the sample pad, containing1%PVP of0.01mol/L PBS (pH7.5). The lowest detection test strip prepared using thistechnology limits as low as0.9ng/mL, eye-measurement were5ng/g and10ng/grespectively in shrimp and fish tissues. Comparied with indirect competitive ELISA, thetest strip can effectively detected positive when the shrimp, fish ENR residues higer than5ng/mL,10ng/mL respectively, while lower than the value was negative.. The test strips canbe stored at room temperature and dry conditions for at least one year.