The purification and development of a quantitative enzyme linked immunosorbent assay (ELISA) for the measurement of vitellogenin in diploid and triploid brook trout (Salvelinus fontinalis)

Abstract

Vitellogenin (VTG) was purified from the plasma of estradiol-17β (E₂) treated male brook trout (Salvelinus fontinalis; bt) using gel filtration and anion exchange chromatography. An antiserum to bt-VTG was raised in rabbits, then used to detect bt-VTG by Western blot analysis. Purified bt-VTG and its antibody were then used to develop an antibody-capture, competitive enzyme-linked immunosorbant assay (ELISA). Confirmation of the purified protein as bt-VTG was based on the characteristics of high molecular weight (∼562 kDa), dominant plasma protein in vitellogenic females and induction by exogenous E₂ treatment in males. The antiserum recognized a major 184 kDa polypeptide as well as minor bt-VTG degradation products (150–66 kDa) in purified bt-VTG and in plasma from vitellogenic females. Low levels of antibody cross reactivity were shown with plasma from nonvitellogenic females, control rabbit serum, and several other antisera known to not contain VTG. The ELISA had a minimum detection limit of 8 ng ml⁻¹ and intra- and inter-coefficients of variation less than 9% and 15%, respectively. The ELISA demonstrated parallel binding slopes among dilution curves of purified bt-VTG standard and plasma from diploid and triploid brook trout females. Using the ELISA, maximum plasma VTG levels of 93.5±33.6 mg ml⁻¹ were detected in vitellogenic diploid females, whereas only 0.18±0.15 mg ml⁻¹ were detected in triploid females of the same age (n=5 for each). Diploid and triploid males cohabitating with vitellogenic females showed measurable levels of plasma VTG during vitellogenesis in females (i.e., 0.17 and 0.06 mg ml⁻¹, respectively (n=1)).