Optimization and validation of a California halibut environmental estrogen bioassay using a heterologous ELISA

Abstract

Vitellogenin, the estrogen-inducible yolk protein precursor, serves as an indicator of exposure to estrogen mimicking environmental contaminants. An ELISA for the measurement of California halibut plasma vitellogenin was optimized and validated using a commercially-available antibody developed for another flatfish species, turbot. Attempts to enhance assay performance by addition of a biotinylated antibody, polyethylene glycol, and Tween-20, and altering the preincubation step are described. Inclusion of overnight preincubation was critical for low detection limits. Increasing the amount of Tween-20 to 0.05% in buffers was most effective in achieving accurate quantification of spiked plasma samples. At the IC50, the average recovery of spiked plasma samples was 104% and the interplate CV was 12%. The working range of the assay was 33–1000 ng/mL, while the detection limit in a plasma sample is 2.2 μg/mL. The performance of this assay compared very well to a homologous assay demonstrating that commercially-available antibodies can facilitate the development of bioassays for local environmentally-relevant species. The dose response relationship of halibut Vg to the model compounds 17β-estradiol and pnonylphenol show that it is a suitable model for further studies of estrogen mimicking contaminants.