Plasma vitellogenin levels during the annual reproductive cycle of the female rainbow trout (Oncorhynchus mykiss): establishment and validation of an ELISA.
Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric from of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20-320 ng/ml (80-20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.
Bon
E
E
Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Talence, France.
Barbe
U
U
Nuñez Rodriguez
J
J
Cuisset
B
B
Pelissero
C
C
Sumpter
J P
JP
Le Menn
F
F
D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005779:Immunodiffusion; D008297:Male; D008970:Molecular Weight; D017686:Oncorhynchus mykiss / Q000097:blood* / Q000502:physiology; D012098:Reproduction; D012621:Seasons; D014818:Vitellogenesis; D014819:Vitellogenins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification
D014819:Vitellogenins; D004958:Estradiol