Gag (Mycteroperca microlepis) vitellogenin: purification, characterizationand use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper

Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), c an be used as a biochemical indicator of maturation in female fish. Here we report on purification and partial characterization of VTG from a temperat e marine serranid, the gag(Mycteroperca microlepis). Development of a compe titive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is als o described. The gVTG was purified by DEAE-agarose anion exchange chromatog raphy from a pooled plasma sample collected from several juvenile gag after they were injected with 17 beta-estradiol. The protein appeared as a major band of M(r)congruent to 183 000 after SDS-PAGE +/- Western blotting using either a specific rabbit antiserum to gVTG or a universal monoclonal antib ody for vertebrate VTGs. Amino acid composition analysis and N-terminal pep tide sequencing verified that gVTG is similar in primary structure to VTG f rom several other teleost species. The purified gVTG and its specific antis erum were used to develop a sensitive, competitive, antibody-capture ELISA for quantifying the protein in blood plasma from maturing females. VTG leve ls in maturing female gag were highly correlated with oocyte growth and cir culating testosterone and 17 beta-estradiol levels, whereas VTG was non-det ectable in juveniles, immature females or males. Two size-based maturity sc hedules for female gag were constructed, one utilizing detection of VTG in their circulation as a marker of maturity and the other relying on histolog ical evidence that their ovaries were in vitellogenic or later stages of ma turation. The two schedules were virtually identical. The gVTG ELISA was al so used to detect VTG in blood plasma from mature Nassau grouper (Epinephel us striatus) and red hind (E. guttatus). As with gag, the assay was complet ely reliable for discriminating between reproductively mature females versu s males from these two grouper species.