| Neomycin, an aminoglycoside antibiotics produced by Streptomyces fradiae, is widely used in veterinary medicine to treat bacterial infections.Neomycin has a broad-spectrum antibiotic due to its growth inhibition of Gram-positive bacteria and Gram-negative bacteria. Neomycin is potentially ototoxic and nephrotoxic to human and animals, so it is essential to monitor its residues in foods for public health. So it is very necessary to develop a simple and reliable analytical method to monitor neomycin residue levels in the livestock products.Neomycin-KLH, neomycin-BSA, neomycin-OVA and neomycin-HRP conjugates were prepared with EDC methods described by Mahon and Haasnoot. The concentration of the conjugates are 2.39,2.12,3.13 mg/mL respectively determined by ultraviolet radiation. Eight-week-old BALB/c mice were immunized with Neomycin-KLH and neomycin-BSA respectively. Spleen cells from immunized mice were fused with myeloma cells (Sp2/0) using PEG 1500.Competitive indirect ELISA was used for screening clones and subclone-confirm. Three cell lines named 11B5,3B1,10H7 were obtained. The result of the indirect competitive ELISA shows that 11B5 was comfirmed to be specifically against Neomycin, the IC50 was 2ng/mL.Reliable analytical methods are required to monitor neomycin residue levels in the livestock products.In particular, a more simple and rapid detection method is required in the veterinary fields.Competitive direct ELISA was developed using monoclonal antibody to detect neomycin in milk. No cross-reactivities of the antibody was observed with other aminoglycosides(kanamycin, gentamycin, streptomycin, amikacin, tobramycin) based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 29.2ng/mL in milk. Recoveries of neomycin from spiked milk samples ranged from 68～94%.The developed ELISA is reliable with good correlations (R2=0.9949).
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