Preparation and Immunological Properties of Neomycin Monoclonal Antibody

Neomycin was conjugated to carrier protein BSA and OVA by the method of EDC to form the immunizing antigen BSA-NEO and the coating antigen OVA-NEO.IR and SDS-PAGE were used to identify the NEO artificial antigen.Balb/c mice were immunized with BSA-NEO.The titre of polyclonal antibody was detected by indirect ELISA and blocking ELISA.The hybridoma lines that secrete NEO mAb were established with monoclonal antibody hybridoma technology.The immunological traits such as titer,affinity,sensitivity and specificity of the mAb were characterized.The results showed that NEO artificial antigen was synthesized successfully.Three hybridoma cell lines of 1E9、4E8 and 1G1 were screened for specificity to NEO.The indirect ELISA titer of the mAb were 1∶2.56×103,1∶1.28×103 and 1∶5.12×103 in supernatant,and 1∶5.12×105,1∶2.56×105 and 1∶1.02×106 in ascites.The affinity constant(Ka)of 1G1 was about 3.75×1010(L/mol).The mAb of 1G1 showed good sensitivity with an IC50 of 2.57 ng/mL to NEO.No cross-reactivity to other compounds was detected,snch as Gentamicin,Streptomycin,Oxytetracycline,Ciprofloxacin,Difloxacin,etc.The NEOmAb with high-titer,sensitivity and specificity had been generated,and could be used to establish the immunoassay of NEO residues in feed and animal food.