A Simple Defatting Method Using a Partition Method of Acetonitorile and n-Hexan for Radioimmunoassay of Low Blood Levels of Estradiol-17β

Abstract

A convenient and simple defatting method using acetonitorile and n-hexan for estradiol-17β radioimmunoassay (RIA) is established. Using the present defatting method, plasma or serum estradiol-17β concentrations of various animals having low blood levels of estradiol-17β can be measured conveniently without the purification of estradiol-17β using LH20 column chromatography. Data of plasma from cows used the present defatting method were coincided well with data by defatting method using methanol and n-hexan. The specific steps in the procedure of this defatting methods were as follows. 1) Ether extraction: Standard solutions or plasma (serum) samples (1 ml) were transferred to glass tubes (13 × 100 mm) for ether extraction. Three ml diethyl ether was added to each tube. The tubes were agitated to extract estradiol-17β and stilled for 5 minutes. The tubes were subsequently dipped in dry ice-ethanol bath to freeze the water layer and ether layer was transferred to a new glass tube (12 × 75 mm) by decapitation. 2) Defatting: To remove substances that interfere with the estradiol assay, after drying the ether, a solution of a mixture of 1 ml n-hexane and 0.5 ml acetonitorile (V/V) was added, and the tubes were mixed. This procedure was repeated twice. After aspiration of the hexane phase, the methanol phase was dried with nitrogen gas and redissolved in 0.1 ml PBS (50 mmol/L, pH 7.5) containing 1% (W/V) BSA (BSA-PBS), and the solution was used for the estradiol RIA.