Enzyme-Linked Immunosorbent Assay (ELISA) of Vitellogenin in Temperate Basses (GenusMorone): Plasma and In Vitro Analyses

Abstract

Blood levels of the egg yolk precursor vitellogenin (VTG) can be used as a definitive marker for the onset and progress of maturation in female teleosts. In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed to measure VTG in blood plasma from three species of temperate basses. The antigen capture, competitive ELISA is based on a rabbit antiserum raised against striped bass Morone saxatilis VTG and uses purified striped bass VTG as standard and in the final antigen capture step. The assay was validated for detecting VTG in the plasma of maturing female striped bass, white perch M. americana and white bass M. chrysops. Serial dilutions of blood plasma from vitellogenic females of all three species yielded VTG curves that paralleled the standard curve in the ELISA, whereas no cross reactivity was observed for plasma obtained from males of any Morone species. The working range of the ELISA was 33–1,118 ng/mL (90–10% of binding), and the intra- and interassay coefficients of variation (100 × SD/mean) at 50% binding were 3.8% (N = 20) and 5.94% (N = 4), respectively. Complete recovery (detection) in the ELISA was verified for a known quantity of VTG added to male striped bass plasma. Changes in plasma VTG concentrations during the annual reproductive cycle of female striped bass were measured both by ELISA and an established radial immunodiffusion assay (RIDA) based on the same antiserum and standard. Vitellogenin was detected in maturing females 7–8 months prior to spawning and the correlation between individual VTG values measured by ELISA and the RIDA was very high (r ² = 0.95). The highly sensitive and precise VTG ELISA should allow aquaculture and fisheries biologists to evaluate the gender and maturational status of individual fish of any Morone species during most of the year. Finally, VTG was detected by ELISA in incubation medium following culture of white perch liver fragments with 1 × 10⁻⁶ M estradiol-17β, providing the basis for an in vitro method to study the physiology and toxicology of vitellogenesis in temperate basses.