Monoclonal Antibody Enzyme-Linked Immunosorbent Assay to Quantify Vitellogenin for Studies on Environmental Estrogens in the Rainbow Trout (Oncorhynchus Mykiss)

Abstract

Vitellogenin (VTG) induction has proved to be a valuable biomarker for assessing exposure to environmental estrogens in fish. The widespread use of VTG in this regard has lead to the need for standardized assays to quantify VTG, and monoclonal antibodies have the potential to help accomplish this. A VTG enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody prepared against Atlantic salmon (Salmo salar) VTG (MAb BN-5) and its ability to quantify VTG in the rainbow trout (Oncorhynchus mykiss) compared with a rainbow trout vitellogenin (rt-VTG) ELISA that employed homologous polyclonal antibodies (PAb). In routine protocols, the working range of the homologous rt-PAb VTG ELISA was between 9 ng/ml and 70 ng/ml (80- 20% relative maximum binding [B/Bo]) with a 50% B/Bo of 25+/-0.9 ng/ml and inter- and intraassay variations at 50% B/Bo of 7% (n = 7) and 8% (n = 15), respectively. The working range of the MAb BN-5 VTG ELISA was between 60 ng/ml and 850 ng/ml (80-20% B/Bo) with a 50% B/Bo of 227+/-22 ng/ml and inter- and intraassay variations at 50% B/Bo of 5% (n = 10) and 9% (n = 12), respectively. In the routine protocols, detection limits for measurement of plasma VTG in rainbow trout (at 80% B/Bo; and given the requirement to dilute plasma to a minimum of 1:10 for the assays) were 90 ng/ml for the polyclonal rt-VTG assay and approximately 600 ng/ml in the monoclonal antibody assay. In juvenile female rainbow trout exposed to a series of doses of estradiol-17beta (E2) and 4-tert nonylphenol (4-NP), there were no differences in the vitellogenic responses measured in the PAb and MAb BN-5 VTG ELISAs. The monoclonal MAb BN-5 VTG ELISA is likely to be of considerable value for studies on environmental estrogens in juvenile female rainbow trout in standardized tests.

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

“…[7]), and the inherently unstable nature of the Vtg protein, which is designed to disintegrate into smaller yolk peptides after uptake into the oocyte (see [2]). Furthermore, the small size of the selected fish test species creates methodological challenges with respect to isolation of Vtg for standard preparation and sample handling.…”

“…A competitive ELISA for rainbow trout (Oncorhynchus mykiss) using a monoclonal antibody to salmon Vtg, resulted in an assay with a working range between 60 and 850 ng Vtg/mL [7]. Although better sensitivity in competitive assays has been obtained with polyclonal antibodies for both rainbow trout (ibid.…”

“…Plasma Vtg can vary maybe 100 million-fold, from a few ng/mL in unexposed male fish, to the 50-150 mg/mL or above found in estrogenized salmonids (e.g. [7]). To be able to quantify this enormous range in blind samples, the working range of the assay should preferentially be as wide as possible.…”

Carp vitellogenin detection by an optical waveguide lightmode spectroscopy biosensor

“…Therefore, a vitellogenin-free carp serum was first prepared to evaluate this effect. As male fish have the dormant vitellogenin gene(s) and the estrogen receptor(s) necessary for vitellogenin induction, this biomarker protein is normally not present in the blood of male fish (Tyler et al, 2002). Based on this fact, we prepared the blood sera from three male carp and analyzed the presence of vitellogenin by Western blotting.…”

“…It is normally present only in the blood of female fish, but males also have vitellogenin gene(s) and the estrogen receptors(s) necessary for vitellogenin regulation (Tyler et al, 2002). Therefore, the exposure of female and male fish to endocrine disruptors such as 17␤-ethinylestradiol (the contraceptive pill), dioxins and PCBs can trigger vitellogenin gene expression, and as a consequence, vitellogenin accumulates in the blood abnormally (Chen, 1983;Tyler et al, 1996).…”