| In this paper,the direct competitive enzyme-linked immunosorbent assay(ELISA) method for the determination of neomycin residues in food products had been developed. The method of coupling the hapten to carrier albumens(keyhole limpet hemocyanin,KLH) was investigated.The anti-neomycin antibody produced was specific for neomycin.The method of coupling the hapten to horseradish peroxidase(HRP) was investigated.The sensitivity of the method was higher,and the limit of detection(LOD) of neomycin residues in different matrix was 0.1±0.01μg/kg.The assay precision were investigated and the variations of inter-assay and intra-assay reproducibilities were all lower than 20%.The effect of different matrices on the ELISA methods were investigated detailedly,the matrix removal methods for different samples and the simple extraction procedures were optimized.A series of animal-derived food samples such as milk,chicken muscle,pig muscle,fish,kidney and egg could be analyzed directly by the direct competitive ELISA after dilution in 0.5%BSA/PBS.The LOD of neomycin residues in six samples were all lower than maximum residue limit(MRL 500μg/kg orμg/L).The spiked experiments of neomycin in six samples were investigated,and the recoveries were all between 75～105% which could satisfy the need of fast screening assays.Because neomycin have no UV or visible absorption,the derivative reaction was studied. The o-phthaldialdehyde(OPA) was selected.The conditions of the experiments of the derivative reaction were investigated.In this paper the HPLC method was applied,which could validate the direct competitive ELISA method.Sample extracts were analyzed by the ELISA and the HPLC,and results obtained by the two methods correlated well,the correlation was greater than 0.9.The stability experiments of the neomycin kit showed that the detection ability could stand after 6 months at 4℃.This research will be useful for the development of anti-neomycin ELISA kit.
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