Development of a highly sensitive, second antibody format chemiluminescence enzyme immunoassay for the determination of 17β-estradiol in wastewater

Abstract

In the present work, a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) method for the determination of 17β-estradiol (E2) in the wastewater was developed, which used the fluorescein- iso-thiocyanate (FITC)–anti-FITC system as solid phase and the biotin–streptavidin system as signal amplification system, with the highly sensitive 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD)–alkaline phosphatase (ALP) system as chemiluminescence detection system. The specific polyclonal antibody was produced against a conjugate of estradiol–bovine serum albumin (BSA), biotinoyl–diaminodioxaoctane–estradiol conjugate was synthesized. The specificity of anti-E2 antibody was also studied. The results showed that anti-E2 antibody has no cross-reactions for estriol, ethinyl E2, estrone commonly coexisting with E2 in the wastewater. Samples were prepared with solid-phase extraction using C 18 cartridges for the removal of matrix effects. The linear range was 2.5–1600 pg ml −1 and detection limit was 1.5 pg ml −1. As our known of knowledge, the method was more sensitive for the determination of E2 in water compared with other immunoassay methods. Recoveries were in the range of 80–110% in spiked tap water and wastewater. This method has been successfully used for the detection of E2 in wastewater, compared with the commercially radioimmunoassay (RIA) kit, the correlation was good.