Establishment Of ELISA Rapid Test Method Of Sarafloxacin Of Animal Origin Food

Sarafloxacin (SARA) is one kind of new-type veterinary drugs, which was marketed in the US in1995. SARA is the first kind of fluoroquinolones approved in the United States for animal-derived products;it is an excellent antibacterial agent with wide spectra, good sterilization and low price, and thereforewidely used.SARA is approved for the treatment of animal diseases caused by pathogenic bacteria such asEscherichia coli, but if fluoroquinolones residues in the animal-derived products may contribute toincreasing antibiotics resistance of human pathogens. According to the rules and regulations in our country,the Maximum Residue Limit (MRL) of SARA should be no more than80μg/Kg. The current detectionmethods for fluoroquinolones include Fluorescence spectrophotometry, HPLC, solid-phase extraction andso on. The above methods have the high accuracy and sensitivity, but with several disadvantages and limits,such as more expensive equipment, the cumbersome sample pre-treatment, only limited to use in thelaboratory, which cannot satisfy the field fast detection for its inconvenience. Therefore, it is necessary toestablish a new fast detection method for SARA residues with high accuracy and low cost. Enzyme-linkedImmunosorbent Assay (ELISA) is one kind of fast detection methods with some advantages, such as easyoperation, fast detection, high accuracy, and high sensitivity, satisfy the field fast detection, low cost and soon, therefore they could be widely used in occasion inconvenient for large-scale instruments mentionedabove. With the continuous development of ELISA in recent years, ELISA has been abroad applied in theaspect of rapid detection methods for pesticides and veterinary drugs.To establish ELISA detection method for SARA, the monoclonal antibodies should be prepared.SARA is one kind of micromolecules and belongs to hapten that only has reactionogenicity but noimmunogenicity. SARA needs to be combined with macromolecules to form artificially synthesizedimmunogen and then obtain immunogenicity. Mice were immunized with artificial antigens, polyclonalantibodies from the antiserum were used to prepare monoclonal antibodies of SARA, and then finallyELISA detection method for SARA was established. This study, based on the characteristics andadvantages of ELISA in the detection, via synthesizing immunogen artificially, immunizing mice to obtainpolyclonal antibodies with high affinity and selectivity, lays a foundation for preparing monoclonalantibodies of SARA and establishing ELISA detection method for SARA. The main contents and results of this study were as follows.1. Synthesis and identification of the artificial antigenSARA-BSA and SARA-OVA were prepared by conjugation of SARA with BSA and OVArespectively via carbodiimide method (NHS method). The characterization of hapten-protein conjugateswere by two methods, ultraviolet spectrophotometry (UV) and sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) respectively. The results elementarily showed that the artificial antigen ofSARA have been synthesized successful.2. Preparation and identification of monoclonal antibodiesThe titer and sensitivity of polyclonal antibody were detected to select the mice in cell fusion. Viausing monoclonal antibody technique and ELISA screening system, two hybridoma cell strains which canproduce anti-SARA monoclonal antibodies steadily were selected and named as1G3and3H3. Massiveascites were induced from in vivo method, after being purified and identified, the titer of antibody in theascites was5.12×105，IC50=6.34ng/mL, and the affinity constant(Ka) was8.55×108L/mol; The rate of crossreaction between SARAmAb and norfloxacin was1.06%, and all the rates of cross reaction to othercompounds were no more than0.5%.3. Assembly of the competitive ELISA kit for rapid detection of SARAVia using prepared monoclonal antibodies and indirect competitive ELISA detection principle,competitive ELISA kit for SARA residual detection was assembled. The calibration curve of SARA-Kitwith standard SARA inhibitor was typical sigmoid curve, the linear regression equation wasy=-0.3848x+0.7363, R2=0.9901, IC50=4.11ng/mL. The SARA-Kit has high specificity, and the rates ofcross reaction to other compounds were lower than1%, and the detection limit for SARA was2.06ng/mL.When2,10,50,100ng/mL SARA were spiked into Chicken liver and Chicken, the averagerecoveries were85.3%and88.7%respectively, the average variation coefficients in the same batch andbetween batches were both lower than15%. The validity of SARA-Kit in4℃was above180days. TheSARA-Kit has many advantages such as high specificity, high repeatability, high sensitivity, high accuracy,high veracity and stability, which can fully satisfy the needs of rapid detection for SARA residuals.