Enzyme-linked immunosorbent assay for the detection of neomycin in milk: effect of hapten heterology on assay sensitivity

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin was developed. Three conjugates were used as immunogens to immunize rabbits. Titers and IC50 values of antisera from various bleeds of rabbits were monitored to confirm final-bleeding time. Final antisera were then screened by competitive indirect ELISA (ciELISA) using homologous and heterologous coating antigen. Thereafter, the effect of different types of hapten heterology on ciELISA sensitivity was evaluated. The results showed that the degree of hapten heterology parallels the improvement of ciELISA sensitivity. The optimized ELISA gave an IC50 value of 0.098 ng/ml, and it showed negligible cross-reactivity with other aminoglycosides. The recoveries of neomycin from spiked milk at levels of 2.5-20 ng/ml ranged from 87 to 109%, and the detection limit was 0.69 ng/ml in milk.