Vitellogenin assay

Reference should be made to the vitellogenin assay that has been used as a biomarker for estrogenic effects in fish (Section 7.6.1). 

Isidori M, Cangiano M, Palermo FA et al (2010) E-screen and vitellogenin assay for the detection of the estrogenic activity of alkylphenols and trace elements. Comp Biochem Physiol C ... 

Petty et al. (1998, 2000) used a vitellogenin (VGT) assay to assess the endocrine disrupting potential of contaminants in purified SPMD extracts. VGT is an egg yolk phosphoprotein precursor that is synthesized in the liver of female teleosts in response to estrogen from the ovary (Bailey, 1957). A wide variety of environmental contaminants have been shown to have estrogenic activity (Colborn et al., 1993). Equal portions of purified extracts from SPMDs, exposed in the Missouri River after the flood of 1993 and from the IWWTP at the Nogales Wash deployment were individually injected into immature rainbow trout (Oncorhynchus mykiss) as described in Section 6.4. The SPMD extracts contained elevated levels of complex mixtures of contaminants, including PAHs and pesticides. The fish injected with these sample extracts exhibited VGT induction, while no induction was observed in fish injected with any of the blank sample extracts. 

Several assays, such as the ultra-sensitive luminescent ELRA developed by Seifert (2004) with a detection limit of 20 ng L-1 for 17 (3-estradiol, have been reported in the literature. Although these assays are simple to use, many factors, such as differences in culture conditions, cell density or cell-line clones, affect the potency of estrogenic substances, and that makes the standardization of these methods difficult. The induction of several proteins or enzyme activities (e.g. the increasing levels of alkaline phosphatase, cathepsin D, prolactin and vitellogenin as a consequence of progestogens) has also been used to study estrogenicity. However, expression of these proteins or enzyme activities is restricted to specific cell lines and cannot be extrapolated to other tissues or species. 

Fenske,M., R. van Aerie, S. Brack, C.R. Tyler and H. Segner. Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals. Comp. Biochem. Physiol. 129C 217-232, 2001. 

Assessment of the estrogenic impacts of EDCs requires development of sensitive, simple, and accurate assay systems for evaluating their bioactivity. Several estrogen-regulated proteins have been utilized as biomarkers of exposure of fish and wildlife to EDCs. The yolk precursor protein, vitellogenin (Vg), has been used most frequently for such assessments. In oviparous vertebrates, Vg is produced by the liver of maturing females in response to E2, secreted into the bloodstream, and then taken up... 

Heppell, S.A., N.D. Denslow, L.C. Folmar and C.V. Sullivan. Universal assay of vitellogenin as a biomarker for environmental estrogens. Environ. Health Perspect. 103 9—15, 1995. 

Heppell, S.A., L.F. Jackson, G.M. Weber and C.V. Sullivan. Enzyme-linked immunosorbent assay (ELISA) of vitellogenin in temperate basses (Genus Morone) plasma and in vitro analysis. Trans. Am. Fish. Soc. 128 532-541, 1999. 

Heppell, S.A. and C.V. Sullivan. Gag (Mycteroperca microlepis) vitellogenin purification, characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper. Fish Physiol. Biochem. 20 361—374, 1999. 

Kwon, H.C., Y. Mugiya, J. Yamada and A. Hara. Enzyme linked-immunosorbent assay (ELISA) of vitellogenin in white-spotted charr, Salvelinus leucomaenis. Bull. Fac. Fish. Elokkaido Univ. 41 241-259, 1990. 

Ohkubo, N., K. Mochida, S. Adachi, A. Hara, K. Hotta, Y. Nakamura and T. Matsubara. Development of enzyme-linked immunosorbent assays (ELISAs) for two forms of vitellogenin in Japanese common goby (Acanthogobius flavimanus). Gen. Comp. Endocrinol. 131 353-364, 2003. 

Fig, 2. Ouchterlony radial immunodiffusion assay of sera and tissues from intact and estrogen-treated female sirtalis. Center well assay A rabbit 337 anti-vitellogenin. Center well assay B vitellogenin, 100 mg/ml. Wells 1 and 4 both assays estrogen-treated female serum. Well 2 both assays untreated female serum. Well 3 both assays dermal tissue from untreated female. Well 5 both assays dermal tissue from untreated female, treated in vitro with estradiol in PBS. Well 6 both assays dermal tissue from the same untreated female as the serum in well 2 and the tissue in well 5, treated as in well 5 but without estradion. Drawn from a plate in Garstka 0-982). 

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