Synthesis And Identification Of Neomycin Artificial Antigen

Neomycin is an aminoglycoside antibiotic which was widely used in veterinary medicine because of its broad-spectrum antibacterial effect. However, despite its impressive clinical successes, neomycin is potentially ototoxic and nephrotoxic to human and animals, and it threatens the safety of livestock products so that many countries forbid or restrict the use of neomycin. Thus monitoring of its residues in foods is essential for the maintenance of public health. There are three major kinds of methods for the detection of neomycin residues in food:physicochemical method, microbioassay and immunological method.Enzyme-linked immunosorbent assay has a lot of advantages such as convenience, rapid, sensitive, special and so on. It becomes the major method for the detection of veterinary drug residues in food. A perfect artifical antigen is the prerequisite of the development of fast ELISA.In this study we prepared artificial antigen of NEO, identified it by physicochemical method and evaluated its immune effect. According to the protein coupling theory, NEO was conjugated to carrier protein bovine serum albumin (BSA) and ovalbumin(OVA) by the method of EDC to form the immunizing antigen NEO-BSA and the coating antigen NEO-OVA. The concentration of the conjugates was determined by ultraviolet radiation. IR and SDS-PAGE were used to identify NEO artificial antigen. BALB/c mice were immunized with NEO-BSA, the titer of polyclonal antibody (pAb) was detected by indirect ELISA, the sensitivity of pAb was identified by blocking ELISA and the specificity of pAb was identified by the method of cross-reaction test.The results showed that the concentration of NEO-BSA and NEO-OVA are6.344,4.122mg/ml respectively determined by ultraviolet radiation. IR spectrogram of NEO-BSA was changed when compared to BSA, the electrophoresis strip of NEO-BSA lagged behind BSA and appeared the visible trail when compared to BSA. pAb against NEO were produced by immunizing BALB/c mice with NEO-BSA conjugate. We established an indirect ELISA method to detect neomycin antibody and got the antiserum titer of1:3200, IC50of105μg/ml. The cross-reactivity rate of the antibody with gentamicin, kanamycin and streptomycin were below0.5%, while the cross-reactivity rate of the antibody with salbutamol and terramycin were below0.1%, indicating that the antibody is highly specific for neomycin. This research will be useful for the development of anti-neomycin monoclonal antibody, and lay a foundation for the development of rapid diagnostic kit of neomycin.