Gilthead seabream (Sparus aurata) vitellogenin: purification, partial characterization, and validation of an enzyme-linked immunosorbent assay (ELISA).
VTG was purified from seabream Sparus aurata plasma by ion exchange chromatography on a DEAE-Sepharose column. The vitellogenin was characterized and its properties were determined. The molecular mass of the native form, obtained by Sephadex G-200 column, was around 450 kDa, whereas an apparent molecular mass of 180 kDa was detected by electrophoresis under denaturing and reducing conditions, suggesting a dimeric form for the native protein. The presence of carbohydrates was determined using concanavalin A, while the presence of phosphate groups was detected by Stains-all, a cationic stain. These data together with the sex specificity, the estrogen inducibility, and the cross-reactivity of the abVTG against the major yolk proteins identifies this protein as vitellogenin. The validated ELISA was used for a rapid and reliable measurement of plasma VTG changes related with those of estradiol-17beta in female broodstock.
Mosconi
G
G
Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, Italy.
Carnevali
O
O
Carletta
R
R
Nabissi
M
M
Polzonetti-Magni
A M
AM
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D004958:Estradiol / Q000097:blood; D008099:Liver / Q000187:drug effects; D008297:Male; D010473:Perciformes / Q000097:blood / Q000502:physiology*; D015203:Reproducibility of Results; D014819:Vitellogenins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*
D014819:Vitellogenins; D004958:Estradiol