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)

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