Direct enzyme immunoassay of fecal estrone derivatives in dairy cows

Abstract

ABSTRACTThe present study was undertaken to develop a novel, practical and simple procedure for enzyme‐linked immunosorbent assay of fecal estrone derivatives (estrone, estrone sulfate and estrone glucuronide) in dairy cows. Fecal solution, prepared by mixing feces with borate buffer, was applied directly to wells without extraction, and incubated with anti‐estrone antibody and horseradish peroxidase‐labeled estrone. Estrone sulfate was used as the standard. The sensitivity of the assay was estimated as 0.15 ng/mL (0.6 ng/g). The intra‐assay and inter‐assay coefficients of variation were 5.3–8.1% and 13.4–15.7%, respectively. The recovery rate was 78–102%. Only 4 h were needed to complete an assay to measure fecal estrone derivative concentrations. A significant positive correlation was established between plasma estrone sulfate concentrations and fecal estrone sulfate equivalent concentrations. When fecal estrone sulfate equivalent concentrations were measured in pregnant dairy cows, a gradual increase from day 150 of pregnancy, and subsequent drastic increases from day 240 to calving date were observed. These results suggest that the direct enzyme immunoassay procedure developed in the present study is a practical and reliable method for measuring fecal estrone derivative concentrations.