Arctic charr (Salvelinus alpinus) vitellogenin: development and validationof an enzyme-linked immunosorbent assay

Vitellogenin (Vtg) was isolated from plasma of estradiol-17 beta-treated Ar ctic charr males by double precipitation with MgCl2-EDTA and distilled wate r, followed by ion-exchange chromatography. The monomeric form of Vtg, as r evealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 1 58 kDa. The purified Vtg was used to raise a polyclonal antibody for Vtg (A bVtg), and the specificity of the AbVtg was assessed by Western blot analys is. No cross-reactivity was observed with plasma from control males. Using this AbVtg, a competitive enzyme-linked immunosorbent assay was developed. The detection limit of the assay was 2 ng ml(-1), and the intra- and inter- assay variations determined from plasma samples were 8.6 and 13.3%, respect ively. The assay was validated by quantification of Vtg in plasma samples o btained during a reproductive cycle of Arctic charr. Vtg of females increas ed gradually from 3 mg ml(-1) in early March to a peak value of 22 mg ml(-1 ) in late August, followed by a rapid drop to 2 mg ml(-1) at the time of sp awning in mid-October. The temporal changes in plasma Vtg of females correl ate well with the reproductive cycle. Vtg was undetectable in males, except on some sampling dates during July-September when minute amounts (3-13 mu g ml(-1)) were detected in some individuals. (C) 1999 Elsevier Science Inc. All rights reserved.