Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity.
Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl₂/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2-787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.
Pêches et Océans Canada, Institut Maurice-Lamontage, 850 route de la Mer, Mont-Joli, QC, G5H 3Z4, Canada, domynick.maltais@dfo-mpo.gc.ca.
Domynick
D
Maltais
Bernard-Antonin
BA
Dupont-Cyr
Robert L
RL
Roy
Nathalie R
NR
Le François
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008297:Male; D010473:Perciformes / Q000097:blood*; D055550:Protein Stability; D012732:Sex Determination Analysis; D012741:Sexual Maturation; D013048:Specimen Handling; D014819:Vitellogenins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D014819:Vitellogenins