Preparation Of Roxarsone Complete Antigen And Monoclonal Antibodies Against Roxarsone

Roxarsone（ROX）is a kind of organic arsenic preparation,which is widely used in livestock and poultry farming due to its anti-coccidial and growth-promoting effects.However,with the development of science and technology,people gradually realized the adverse effects brought by ROX.,The EU,the United States,China all banned the use of ROX or specified the minimum detection limit of ROX.Therefore,it is necessary to establish a rapid detection method for ROX.Currently,the commonly method that is used to detect ROX is still instrument detection method.Instrument method includ chromatography and mass spectrometry,but these methods require expensive equipment and professional operations.The rapid detection method of immunology has the characteristics of rapid,specific.Also it can detcet large-scale detection samples in a short time.So it is of great significance for rapidly detecting ROX on-site.Antibodies with high specificity and affinity are the key to establishing rapid immunoassay methods.Currently,the rapid immunoassay method for ROX uses polyclonal antibodies as detection antibodies,and there are no reports about ROX monoclonal antibody detection products so far.This study aims to produce highly specific and sensitive antibodies,that can lay the foundation for establishing rapidly immunology detection methods for ROX.In this study,the structural analog of ROX,3-amino-4-hydroxybenzoic acid（HAPA）,was used as a substitute for ROX.BSA and HAPA were linked to prepare artificial synthetic antigen HAPA-BSA through amide bond with carbodiimide（EDC）.The primary amino group of OVA and HAPA were linked to prepare a coating antigen HAPA-OVA.It will be used for determining serum titer.The HAPA-BSA and HAPA-OVA were identified by UV scanning and SDS-PAGE.The results showed that HAPA was successfully linked to BSA and OVA.Three mice were immunized with HAPA-BSA,and serum was collected after four times immunization.The titer of serum was 1:6.4×10³ and the IC₅₀ was 63.533μg/mL.Hybridoma cells were prepared through cell fusion technique,and positive cells were subcloned through limiting dilution method.A hybridoma cell line stably secreting anti-ROX antibody was screened and named as 3G12.The culture medium of 3G12 cell strain was collected.The ELISA results showd the titer was 1:5.12×10²,and the IC₅₀ was 3.147μg/mL.Subsequently,monoclonal antibody was prepared through injecting cells to intraperitoneal to producing ascites.The ascites was collected and purified by octanoic acid-ammonium sulfate method.SDS-PAGE results showed that the band of purified ascites was single after purification.Indirect ELISA was used to determine the ascites titer\,and the results showed ascites titer can reach 1:1.024×10⁵ before and after purification,and the IC₅₀ of ascites is 1.433μg/mL after purification.In summary,this study successfully developed a monoclonal antibody against ROX,which laid the foundation for establishing rapid immunoassay for ROX.