The detection limit of the competitive ELISA is defined as the R-VTG concentration corresponding to 95% antibody binding. Intra-assay variations were evaluated by adding three different plasma samples onto one plate, 12 wells for each sample. Inter-assay variations were evaluated by measuring three different plasma samples in triplicate for eight consecutive days. The coefficient of variation (CV, %) was calculated as the SD divided by the average and multiplied by 100.