Development Of The Rapid Immunoassay Method For The Detection Of Sparfloxacin In Animal Food

Sparfloxacin (SPFX), a third-generation quinolone antimicrobialdrug, is widely used in the treatment of urinary tract infections such as primaryrespiratory, urinary tract, intestinal, biliary tract, skin and soft tissue infections due toits excellent activity against various bacteria and good absorption on oraladministration, it can also be used for aquaculture growth-promoting agent. However,SPFX is easy to accumulate residue in animal organs, and even endanger seriouslyaffect human health, including decreased appetite, vomiting, special sense adverseevents and so on. Now, both at home and abroad have developed an effectivedetection method to control the abuse of SPFX, but the traditional detection methodsresearch is lagging behind, so it is a stringent task to found a simple, convenient,effective and quick technology to detect SPFX.In this study, Based on the analysis of molecular structure and immunogenicityof SPFX, the technology of monoclonal antibodies production was applied toassemble rapid ELISA kit for SPFX residual detection. The main contents and resultsof this study were as follows:1. Synthesis and identification of the artificial antigen for SparfloxacinAccording to chemical structure and physicochemical properties of SPFX, it wascoupled with carrier protein bovine serum abumin (BSA) and ovalbumin (OVA) toprepare immunizing antigen SPFX-BSA and coating antigen SPFX-OVA by themethod of mixed anhydride method and the DCC method, Both UV scanningspectrum and SDS-PAGE were used to identify SPFX artificial antigen, The result ofUV spectrum show that the largest artificial antigen absorption peak migrationoccurred, SDS-PAGE results show that the BSA’s swimming faster than SPFX-BSA,the SPFX-BSA molecular weight greater than BSA,This further shows thatSPFX-BSA has been coupled with success.2. Preparation of monoclonal antibodies and immunological characterization ofSparflxacinOne BALB/C mouse was chosen from four BALB/C mice immunized with SPFX-BSA for cell fusion by the identification of Serum testing, The hybridoma linesthat fused with NS0myeloma cells and The lymphocytes from Balb/c mouse spleenroutinely by using monoclonal antibody hybridoma technology. One sensitivity,specificity of hybridoma cell lines of4H4C8were screened by using detection,screening and clone, the McAb of4H4C8showed good sensitivity with an IC50of27.63ng/mL, little or no cress-reactivity to other compounds. SPFX mAb obtained canbe used to establish immunoassay of SPFX residues.3. Development of rapid determination of SPFX-KitBased on the enzyme-linked immunosorbent assay principle, a competitiveELISA kit for determination of SPFX (SPFX-Kit) was developed with SPFXmAb. Itcan be used for residues of sparfolxacin in animal foods which can be completedwithin a few minutes. The product is rapid, sensitive, specific, simple, and can bewidely used for the qualitative and semi-quantitative and quantitative detection ofSPFX residues.