A convenient homogeneous enzyme immunoassay for estradiol detection

Abstract A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time‐consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol–reporter enzyme conjugate, was prepared by covalently binding β‐estradiol‐6‐(O‐carboxymethyl)oxime to glucose‐6‐phosphate dehydrogenase (G6PDH) by an N ‐hydroxysuccinimide‐enhanced, carbodiimide‐mediated coupling reaction. The estradiol–G6PDH activity can be repressed up to 46% upon anti‐estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit‐log scale‐up to 6.7 µM estradiol. A detection limit of 11.5 nM in estradiol‐spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.