Establishment Of Immunochromatographic Assay For The Detection Of Enrofloxacin In Shrimp And Shellfish

Enrofloxacin(ENR)is one of the fluoroquinolones.Because of its broadspectrum antibacterial,good bactericidal effect,cheap,and stability,ENR has been widely used in the prevention and treatment of veterinary diseases.The abuse of ENR will cause great harm to the human.Therefore,it is urgent to establish a rapid and sensitive detection method for the monitoring of ENR residues in food.In this study,anti-ENR monoclonal antibody was prepared.Based on the antibody,a rapid and sensitive gold nanoparticles(AuNPs)immunochromatographic assay(ICA)was established for the detection of ENR.The main research methods and conclusions are as follows:(1)The ENR hapten was coupled with bovine serum albumin by carbodiimide method to synthesize the ENR complete antigen and immunize mice.After the fourth immunization and the evaluation of serum,the spleen cells from high-efficiency and high-inhibition positive mice were fused with myeloma cells SP2/0.Three kinds of ENR cell lines(3B4,2B4,and 2C5)were obtained by three subclones.Ascites were prepared after cell lines were amplified and cultured.Three kinds of ENR monoclonal antibodies were obtained after purification.Indirect competitive enzyme linked immunosorbent assay was used to evaluate the obtained antibodies.The half maximal inhibitory concentration of ENR antibodies were 0.13 ng/m L,0.22 ng/m L,and 0.49ng/m L,respectively.The 3B4 with the highest sensitivity was selected for subsequent experiments.(2)3B4 was coupled with AuNPs to prepare AuNPs nanoprobes.Based on the AuNPs nanoprobes,a rapid and sensitive ICA was developed for detection of ENR residues in shrimp and shellfish.Under the optimal conditions,the limit of detection(LOD)of AuNPs-ICA was 80 ng/L.The recovery of the method was 95.18%-102.83% in shrimp with coefficient of variation(CV)<12.85% and 97.79%-103.35% in shellfish with CV<11.82%.Recovery experiments showd that the established method had good reliability and accuracy.The sensitivity of this method for rapid detection of ENR residues in shrimp and shellfish could meet the needs of maximum residue limits of China,and it can be used for primary screening of ENR.In summary,the ENR monoclonal antibodies with high sensitivity was prepared,and an AuNPs-ICA was established for the sensitive and rapid detection of ENR.The study provided providing important technical support for the rapid detection of ENR residues in shrimp and shellfish.