There was no significant binding of PcAb or McAb to untreated male plasma in a series of dilutions suggesting that both antibodies were specific for R-VTG. For experimental samples, it was necessary to test several dilutions in order to be in
the linear range of the assay. Three different plasma samples from E2 treated male rare minnow were diluted to four different concentration ranges, and R-VTG concentrations were determined. R-VTG measurements of all dilutions were within the linear range of the assay (R2 > 0.98) indicating that R-VTG concentrations of plasma could be calculated from any of the plasma dilutions.