Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 beta effects on whole animals and primary-cultured hepatocytes.
Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.
Mitsui
Naoko
N
Towa Kagaku Co., Ltd., Hiroshima 730-0841, Japan.
Tooi
Osamu
O
Kawahara
Akira
A
D000418:Albumins / Q000032:analysis*; D000818:Animals; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D022781:Hepatocytes / Q000737:chemistry* / Q000187:drug effects*; D008297:Male; D014819:Vitellogenins / Q000032:analysis*; D014982:Xenopus laevis
D000418:Albumins; D014819:Vitellogenins; D004958:Estradiol