Arctic charr (Salvelinus alpinus) vitellogenin: development and validation of an enzyme-linked immunosorbent assay.
Vitellogenin (Vtg) was isolated from plasma of estradiol-17 beta-treated Arctic charr males by double precipitation with MgCl2-EDTA and distilled water, followed by ion-exchange chromatography. The monomeric form of Vtg, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 158 kDa. The purified Vtg was used to raise a polyclonal antibody for Vtg (AbVtg), and the specificity of the AbVtg was assessed by Western blot analysis. 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%, respectively. The assay was validated by quantification of Vtg in plasma samples obtained during a reproductive cycle of Arctic charr. Vtg of females increased 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 spawning in mid-October. The temporal changes in plasma Vtg of females correlate well with the reproductive cycle. Vtg was undetectable in males, except on some sampling dates during July-September when minute amounts (3-13 micrograms ml-1) were detected in some individuals.
Johnsen
H K
HK
Norwegian College of Fishery Science, University of Tromsø, Norway. helgej@nfh.uit.no
Tveiten
H
H
Willassen
N P
NP
Arnesen
A M
AM
D000818:Animals; D001769:Blood / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D012621:Seasons; D014337:Trout / Q000502:physiology*; D014819:Vitellogenins / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification
D014819:Vitellogenins; D004958:Estradiol