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

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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.