Study On Enzyme-linked Immunoassay For The Detection Of Gentamicin Residues In Food

In this paper, a reliable enzyme-linked immunosorbent assay method was developed for detecting of gentamycin in eight food samples. Gentamycin connected with keyhole limpet hemocyanin using carbodiimide, which was used as immunogen. A polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Gentamycin connected with horseradish peroxidase using carbodiimide, which was used as enzyme labeled antigen. We established a time-saving and high sensitivity direct competition ELISA detection method, the influence of experimental factors (including antibody incubation concentration, ionic strength, pH, etc.) were studied, the standard curve obtained the highest sensitivity and stability when antibody incubation concentration was0.5μg/well, ionic strength was0.01mol/L PBS, pH8.5. The concentration of gentamycin causing15%inhibition is0.03μg/L,50%inhibition is0.3μg/L. The cross-reactivity against other amino-glycoside antibiotics is all low, except for sisomycin, which has52.5%corss-reactivity.Milk, beef, pork, liver, kidney, chicken, fish, eggs, residues were chosen for detection. Milk:centrifugated, removed fat,7.5%trichloroacetic acid extracted, diluted100-folds. Tissue:7.5%trichloroacetic acid extracted (adding2mL to1g sample), diluted50-folds. Egg:7.5%trichloroacetic acid extracted (adding4mL to1g sample), diluted20-folds. The process didn’t require any organic solvents and pre-treatment, adding three different concentrations of gentamycin. recovery rate was between69%-118%. Limit of detection in milk was3μg/L, in other samples was3μg/kg. Inter-and Intra-variation were less than20%, which meant the method established in this experiment had high accuracy and precision to meet the requirements of rapid detection.The stability of antibody enzyme conjugate were studied by stored in37℃. After3days, the standard curve did not change significantly, showed if antibody and enzyme were stored at4℃for less than6months, they may remain essentially the same detection performance. By comparing with commercial kit, the method established here has higher sensitivity.