Flow injection chemiluminescence immunoassay for 17β-estradiol using an immunoaffinity column

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Abstract
A new flow injection chemiluminescent immunoassay was developed for the detection of 17β-estradiol (E2). The method uses p–iodophenol (PIP) as enhancer and is based on a solid-phase immunoassay format in which an E2–OVA immobilized immunoaffinity column inserted in the flow system is used to trap unbound horseradish peroxidase (HRP)-labeled anti-E2 antibody after an off-line incubation of E2 with HRP-labeled anti-E2 antibody. The trapped enzyme conjugate was detected by injecting substrates to produce an enhanced chemiluminescence (CL) response. The linear range for E2 was 10.0–1,000.0 ng mL−1 with a correlation coefficient of 0.996 and a detection limit of 3.0 ng mL−1. The sampling and chemiluminescence detection time for one sample was 400 s after a pre-incubation procedure of 30 min. Serum samples detected by this method were in good agreement with the results obtained by EIA with E2–biotin.