A direct enzyme-linked immunosorbent assay for hexoestrol residues

Abstract

A simple enzyme-linked immunosorbent assay for the detection of hexoestrol (HES) residue has been developed. The HES derivatives, hexoestrol-mono-ether-butyrate-ethyl (HES-MEBE) and hexoestrol-mono-caroxyl-propyl-ethyl (HES-MCPE) were synthesised to enable the coupling of HES and proteins together. The immunogen HES-MCPE-BSA and enzyme-linked antigen HES-MCPE-HRP were prepared by mixed anhydride methods. After the antibody was obtained by immunisation of New Zealand rabbits, a direct enzyme-linked immunosorbent assay was developed for the determination of HES residue. Several parameters were optimised. Excellent linearity (R 2=0.9905) was demonstrated from 0.01 to 8.1 ng/ml. The 50% inhibition value (IC50) and the limit of detection (LOD) were 0.23 and 0.01 ng/ml, respectively. The specificity of the immunoassay was evaluated by determining cross-reactivity of six hormones (diethylstilbestrol, dienestrol, 19-nortestosterone, medroxyprogesterone, testosterone and estrone), and all cross-reactivity rates were <0.5%. The recoveries from beef and fish ranged from 61 to 102%. The stabilisation of the coated plates was also studied. The ELISA developed was used to detect the residues in chicken muscle tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). This proposed method could be applied to routine residue analysis.