Preparation Of Monoclonal Antibodies Against Lasalocid And Development Of An ELISA Method

Lasalocid(LAS)is a polyether type carrier antibiotic produced by Streptomyces lasaliensis.It is mainly used to improve feed utilization and to prevent chicken Coccidial infection.However,Due to unreasonable use of LAS in the actual production,resulting in LAS residues in milk,eggs and animal tissue.There is a serious hazard to human health.At present,the main methods for the determination of LAS residues are HPLC and LC/MS.However,these traditional instruments require expensive equipment and complex preparation,but also professional and technical personnel to operate.These factors affect the number and accuracy of sample detection.ELISA detection of immunological methods,based on Antigen-antibody specificity principle,with a high degree of specificity and sensitivity.ELISA has the advantages of simple operation and suitable for rapid detection of large quantities of samples.In this paper,LAS antigen was synthesized by active ester method,and then BALB/c mice were immunized with immunogen.Basing on the technology of preparation of monoclonal antibody and ELISA,we establish one stable hybridoma cell lines that secrete monoclonal antibodies against LAS.By using the monoclonal antibody,we established the indirect competitive ELISA method for detecting residual of LAS.1.Synthesis and identification of LAS complete antigens.LAS is a semi antigen molecule containing a carboxyl group.BSA and OVA were used as two protein carriers respectively to couple with LAS.The synthesized antigens were demonstrated to be successful by UV-scanning absorption,SDS-PAGE method and immunological methods.The protein concentrations of LAS-BSA and LAS-OVA were 2.54 mg/mL and 2.42 mg/mL by BCA method.2.Preparation of monoclonal antibody against LAS.Using LAS-BSA as immunogen to immunize BALB/c mice.After five times of immunization serum titer was 1:12800.Using hybridoma technique to fusion spleen cells and myeloma cells.By using the ELISA method and the finite dilution method for screening a strain stably secreting antibodies against LAS.Ascites of monoclonal antibodies were prepared by injecting cells of hybridomas into mice abdomen.The protein concentration of ascites were 33.39 mg/mL.The cross experiment shows that the monoclonal antibody has 100%inhibition rate for LAS,and it has no cross reaction with Monensin sodium salt,Salinomycin Sodium,Maduramicin,Cefalotin Sodium and Streptomycin Sulfate.3.The establishment of ELISA detection method.An ELISA method for LAS was established using ascites.The best dilution concentration of LAS-OVA is 1:400 and the best working concentration of ascites is 1:4000.The standard curve of LAS to antibody-antigen reaction was prepared with the linear equation y = 0.376x-0.2374(R2=0.9914),the linear range was 5-1000 ng/mL.IC50=90.22 ng/mL,LOD=6.04 ng/mL.4.The addition and reclamation test of LAS in the milk.The milk sample was diluted 8 times can basically eliminate matrix interference.In the range from 10 to 1000 ng/mL,the linear equation was y= 0.4111x-0.3591(R2 = 0.9912).ICso=121.96 ng/mL,LOD=13.59 ng/mL.When adding 50,500,800 ng/mL LAS to the milk,the recovery ratio was 81.76%～102.41%.