Establishment And Application Of Immunochromatographic Method For Residue Of Four Banned Fluoroquinolones In Aquatic Products

Fluoroquinolones（FQs）is a type of third-generation synthetic quinolone drug with4-quinolone as the basic structure,inhibiting bacterial DNA synthesis by integrating with topoisomerase II and topoisomerase IV,taking bacteriostatic effect both on gram-negative and gram-positive bacteria.FQs have advantages such as broad-spectrum antibacterial activity,rapid absorption,and inexpensive,which is extensively used on the prevention and treatment of various diseases caused by bacteria,chlamydiae,and mycoplasmas in animal husbandry and aquaculture.However,problems such as environmental pollution and food safety have become more and more serious because farmers do not strictly implement the off-drug period instead of abusing FQs for the past years.Additionally,some farmers mix multiple types of FQs in order to enhance treatment efficacy and benefits,leading to multi-FQs residue in animal-derived food.Due to the adverse reactions that FQs can cause on the human body,China has gradually strengthened supervision on the residue of FQs in animal-derived foods.Currently,there are few immunological methods available for FQs detection.Therefore,it is necessary to find a rapid,efficient,sensitive,and broad-spectrum method to identify FQs.Moreover,the preparation of a high-sensitivity and broad-spectrum monoclonal antibody is the key factor in establishing an immunological multiple detection system.Lomefloxacin（LOM）has the similar basic structure with the most FQs drugs,and it can be used to synthesize immunogens for mouse immunization to obtain monoclonal antibody（m Ab）with broad-spectrum recognition.In this paper,the quantum dot beads（QBs）lateral flow immunoassay（LFIA）based on the m Ab was established for simultaneous detection of four banned fluoroquinolones in yellow croaker,sea bass and snakehead.The main research methods and conclusions are as follows:（1）LOM-BSA and LOM-OVA were synthesized by the carbodiimide method.LOM-BSA was used to immunize mice,and mouse serum was evaluated by ic-ELISA.The mouse with high inhibition rates and positive values were selected for cell fusion,and after three rounds of subcloning and screening,five cell lines were obtained.Ascites fluid was prepared by intraperitoneal injection of mice,and the ascites fluid was purified using the ammonium sulfate precipitation method.Five anti-LOM m Abs were obtained,named L2D7、L2E7、L4C9、L1H10,and L5B6.The sensitivity and specificity of the five m Abs were evaluated by ic-ELISA,and their half maximal inhibitory concentrations(IC₅₀)were 0.215、0.206、0.308、0.201,and 0.408 ng m L^-1respectively.The L2D7 antibody cross-reacted with Norfloxacin（NOR）、Pefloxacin（PEF）、Ofloxacin（OFL）、Enrofloxacin（ENR）、Ciprofloxacin（CIP）、Danofloxacin（DAF）、Fleroxacin（FLE）、Orbifloxacin（ORB）,and Gemifloxacin（GEM）.（2）QBs-LFIA based on anti-LOM m Ab was established for the detection of four banned fluoroquinolones in yellow croaker,sea bass and snakehead,respectively.The optimal parameters of QBs-LFIA were optimized,including the optimal p H of m Ab coupling at 6.0,the optimal amount of m Ab at 6.0μg,the optimal amount of EDC at40μg,the optimal concentration of LOM-BSA on the test line at 0.4 mg m L^-1,the optimal concentration of sheep anti-mouse antibody on the control line at 0.8 mg m L^-1,and the optimal probe addition amount at 1.5μL.Additionally,this study compared the extraction efficiency of five different sample extraction solutions and four different acetic acid-containing extraction solutions,and ultimately selected a mixture of acetic acid/acetonitrile/water as the sample extraction solution.Under the optimum experimental conditions,the IC₅₀ of QBs-LFIA based on anti-LOM m Ab for detecting four fluoroquinolones（LOM、NOR、PEF and OFL）in yellow croaker were 0.361、0.902、0.901,and 0.971μg kg^-1,respectively.In sea bass,the IC₅₀ were 0.515、0.312、0.417,and 1.063μg kg^-1,respectively.In snakehead,the IC₅₀ were 0.761、0.505、1.011and 0.872μg kg^-1,respectively.The stability of this method was verified by an accelerated storage experiment,and the results showed that QBs-LFIA is relatively stable and has a longer commercial shelf life.The accuracy of this method was analyzed by a spiked recovery experiment,with recovery rates of 74%-138%and coefficients of variation of 1.72%-16.48%in yellow croaker,sea bass and snakehead samples.In summary,this study developed a QBs-LFIA based on the anti-LOM m Ab for the rapid quantitative detection of LOM、NOR、PEF,and OFL in yellow croaker,sea bass,snakehead.This method not only saves time and reduces costs,but provides a new method for screening and monitoring the presence of prohibited FQs residues in aquatic products.