Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA.
Male smooth flounder (Pleuronectes putnami) were induced to produce vitellogenin (VTG) by injection of 17beta-estradiol (E2). Anion exchange chromatography of precipitated plasma from E2-injected resulted in a single peak consisting of VTG. Smooth flounder VTG has an approximate molecular mass of approximately 520 kDa, determined by gel filtration with molecular weight standards. Purified VTG was used to develop a homologous enzyme-linked immunosorbent assay (ELISA). The flounder VTG ELISA is an indirect antigen competition assay with a detection limit of 15 ng.ml(-1) and a useful range of 30-950 ng.ml(-1) of diluted sample. Intra- and inter-assay precision (as %CV, n=7) ranged from 1.3% to 6.0% and 5.1%, respectively. The ELISA was evaluated using plasma samples collected from a smooth flounder population captured in the Saint Lawrence Estuary. The ELISA is sensitive enough to differentiate males and non-vitellogenic females from vitellogenic individuals during early vitellogenesis.
Pêches et Océans Canada, Institut Maurice-Lamontage, Mont-Joli, Québec, Canada G5H 3Z4. royro@dfo-mpo.gc.ca
Robert L
RL
Roy
Yves
Y
Morin
Simon C
SC
Courtenay
Paul
P
Robichaud
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005432:Flounder / Q000502:physiology*; D008297:Male; D008970:Molecular Weight; D015203:Reproducibility of Results; D014818:Vitellogenesis; D014819:Vitellogenins / Q000097:blood* / Q000302:isolation & purification*
D014819:Vitellogenins; D004958:Estradiol