Production of antibodies and development of an enzyme-linked immunosorbent assay for 17β-estradiol in milk

The residue of 17 beta-estradiol (E-2) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E-2 residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E-2-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E-2 in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC50) against E-2 of 0.17ng/mL, high cross-reactivity (CR) to E-2 benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093g/L, which was enough sensitive to detect E-2 in milk. In spiked samples (0.5, 1, and 2g/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E-2 residue in milk.