Development of an enzyme‐linked immunosorbent assay for quantifying vitellogenin in Pacific salmon and assessment of field exposure to environmental estrogens

Abstract

A competitive enzyme-linked immunosorbent assay was developed to quantitate vitellogenin (VTG) in plasma and serum of coho (Oncorhynchus kisutch) and chinook (O. tshawytscha) salmon. The working range of the assay was 9 to 313 ng/ml (80-20% binding), with 50% binding at 54 ng/ml. The intra-assay and interassay variations at approximately 50% binding were 8.1% (n = 9) and 9.0% (n = 9), respectively. Dilution curves of plasma or serum from coho and chinook females and estrogen-treated males were parallel to the purified coho VTG standard curve. Male plasma samples could be assayed at a minimum dilution of 1:40 (chinook) or 1:75 (coho) without assay interference because of high sample concentration, whereas minimum acceptable dilutions of male serum samples were 1:200 (chinook) or 1:600 (coho). Identification of proper techniques for preserving VTG integrity in plasma and serum samples showed that VTG from both species was robust; both sample types required no protease inhibitor despite subjection to two freeze-thaw cycles. To test its applicability, this assay was used to measure VTG in out-migrating juvenile chinook that were collected from urban and nonurban areas in Puget Sound, Washington, USA. Results showed a small but significant plasma VTG elevation at two urban sites, suggesting that these juveniles may be exposed to environmental estrogens at an early life stage. Also, wild fish tended to have higher plasma VTG levels than hatchery fish collected in the field. Elevation of mean VTG levels was similar to that previously reported in male English sole from the same area, where both males and females exhibited alterations in timing of spawning.

Vitellogenin induction in caudal fin of guppy (Poecilia reticulata) as a less invasive and sensitive biomarker for environmental estrogens

“…Moreover, the parallelism observed between the Lv standard curve and WBH dilution curves of E 2 -exposed male guppy demonstrated that anti-Lv antibody recognized Lv and Vtg similarly. In addition, the ELISA did not detect positive signal in control male WBH, indicating that this assay was very specific to Vtg 30 , 31 . The above results confirmed that the sandwich ELISA established in the present study could accurately quantify guppy Vtg.…”

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar

“…A quantitative and sensitive EIA was developed from the methods outlined in Peck et al (2011) using a polyclonal, rabbit anti-Atlantic salmon Vtg antibody, AA-1 (Biosense Laboratories AS, Bergen, Norway). Costar, 96-well microtiter plates (#3369, Corning Life Sciences, Tewksbury, MA) were coated with 60 ng/ml Atlantic salmon vitellogenin (#9902, Biosense Laboratories, Bergen, Norway) in 0.05 M Na 2 CO 3 and incubated overnight at 4 • C. The following day, plates were washed with cold phosphate-buffered saline (PBS) with 0.05% Tween-20 (Sigma-Aldrich), pH 7.2 (PBST) and blocked with PBST with 2% milk powder overnight at 4 • C. Samples or standards were mixed with the primary antibody (in PBST with 1% milk powder).…”

“…Costar, 96-well microtiter plates (#3369, Corning Life Sciences, Tewksbury, MA) were coated with 60 ng/ml Atlantic salmon vitellogenin (#9902, Biosense Laboratories, Bergen, Norway) in 0.05 M Na 2 CO 3 and incubated overnight at 4 • C. The following day, plates were washed with cold phosphate-buffered saline (PBS) with 0.05% Tween-20 (Sigma-Aldrich), pH 7.2 (PBST) and blocked with PBST with 2% milk powder overnight at 4 • C. Samples or standards were mixed with the primary antibody (in PBST with 1% milk powder). Samples were diluted a minimum of 1:100 to minimize interference and false-positive readings where Vtg was low (Peck et al, 2011) up to 1:10,000 for samples with high Vtg content. These were mixed at 4 • C overnight on a rotary shaker.…”

Quantification of pharmaceuticals, personal care products, and perfluoroalkyl substances in the marine sediments of Puget Sound, Washington, USA

“…As such, they may elicit toxicological modes of action other than what is expected from their intended use in humans and livestock . Recent studies indicating endocrine disruption in juvenile salmon from Puget Sound and evidence of vitellogenin production, histopathological lesions, and other estrogen‐induced disorders in male English sole from urban bays in Puget Sound may indicate aquatic species are exposed to endocrine disrupting substances such as PPCPs.…”