Preparation Of Monoclonal Antibody Against Ofloxacin And Establishment Of Immunochromatography

Ofloxacin(OFL)is a kind of fluoroquinolones(FQs).FQs have a broad antibacterial spectrum and have inhibitory effects on both Gram-negative and positive bacteria.OFL is widely used in aquaculture,animal husbandry and other breeding industries to prevent and treat infectious diseases of livestock and fish.OFL is resistant to degradation in the body,leading to the problem of its accumulation in the environment and the development of bacterial resistance.In 2015,the Ministry of Agriculture issued a notice prohibiting the use of OFL and other four types of FQs in food animals.However,some companies have illegally added veterinary drugs in aquaculture,and the residue of veterinary drug in aquatic products cannot meet the relative standard of China.Colloidal gold immunochromatography(CGIC)has many advantages,such as fast,simple,cheap,stable and suitable for detecting a variety of samples.It has become an effective method for drug residues,especially on-site screening,and can be applied for the detection of OFL residues in aquatic products to ensure the food safety of aquatic products.In this study,the immunogen and coating antigens of OFL were synthesized by the carbodiimide method.Mice were immunized with the immunogen of OFL,and the spleen cells from the mice with high titer and inhibition ratio tail blood were fused with SP 2/0 myeloma cells.After the fusion,the hybridoma cells went through three"screening-cloning"processes to obtain a total of 3 cell lines 7E11-G2-C12-C9,8H6-G3-A1-B2,and 7E11-G2-C12-A10.The ascites was prepared by the method of inducing ascites tumor in vivo,and the antibody was purified using caprylic acid saturated ammonium sulfate method.The three kinds of antibodies with high-quality were obtained,the names were C9,B2,and A10,respectively.The sensitivity and specificity of the three antibodies were preliminarily evaluated by indirect competitive enzyme-linked immunosorbent assay(ic-ELISA).The half maximal inhibitory concentration(IC₅₀)of ic-ELISA based on the three antibodies were 3.85.?g/kg,6.25?g/kg,and 6.96?g/kg,respectively.Except for cross-reactivity to marbofloxacin,the three antibodies were specific.On this basis,the heterologous coating antigens were further screened by ic-ELISA as the detection antigen,and colloidal gold was used as the signal output carrier to prepare immunochromatographic test strips.The transmission electron microscope(TEM)and ultraviolet-visible spectrophotometer(UV-vis)were used to characterize the colloidal gold and the labeled probe.The relevant parameters affecting the immunochromatographic test strip were optimized,including the p H of the system,the amount of labeling antibody,the amount of probe,the concentration of detected antigen and goat anti-mouse antibody.The reaction kinetic curve was established for obtaining the best detection time.The specificity of the established method was evaluated by 21structural analogs of FQs.The standard curve of this method in 0.1 M phosphate buffer saline(PBS)was y=522.735-872.692logx(R²=0.9983)while the linear range was 0.1-2.5?g/kg with the limit of detection(LOD)at 0.169?g/kg.The pre-treatment has been improved on the basis of the entry-exit inspection and quarantine industry standard SN/T 5122-2019.The standard curve based on the matrix of fish was y=800.612-1118.694logx(R²=0.9623)while the linear range was 2.5-5?g/kg with LOD at 0.254?g/kg.The intra-assay and inter-assay recoveries were 87.5%-126%and 85.8%-123.2%,respectively,and the coefficients of variation were 3.1%-11.6%and 3.5%-12.0%,respectively.The qualitative sensitivity based on the naked eyes was 1?g/kg,and the recovery rate and double-blind detection experiments confirmed that the method had good accuracy.In summary,this study had established a method that could qualitatively and quantitatively detect OFL residues in fish,it had the advantages of sensitivity,specificity,and accuracy.Through simple pre-treatment,it could be applied to the rapid detection of OFL in aquatic products.This method has broad application prospects and is expected to be applied to other food safety on-site screening.