Optimization and validation of a California halibut environmental estrogen bioassay using a heterologous ELISA.
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 microg/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 17beta-estradiol and pnonylphenol show that it is a suitable model for further studies of estrogen mimicking contaminants.
Palumbo
Amanda J
AJ
Department of Environmental Toxicology, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States. ajpalumbo@ucdavis.edu
Koivunen
Marja
M
Tjeerdema
Ronald S
RS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002140:California; D004305:Dose-Response Relationship, Drug; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000097:blood / Q000633:toxicity; D004967:Estrogens / Q000097:blood* / Q000633:toxicity; D005260:Female; D005432:Flounder / Q000097:blood*; D010636:Phenols / Q000097:blood / Q000633:toxicity; D011092:Polyethylene Glycols / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D013997:Time Factors; D014819:Vitellogenins / Q000097:blood / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000097:blood / Q000633:toxicity
D000911:Antibodies, Monoclonal; D004967:Estrogens; D010636:Phenols; D011136:Polysorbates; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D011092:Polyethylene Glycols; D004958:Estradiol; D001710:Biotin