Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC₅₀ value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC₅₀ value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.