Radioimmunoassay for Rat Prolactin

Abstract

The most commonly used method for measuring prolactin in biological samples has been the pigeon crop bioassay of Lyons (1). It is possible with this method to estimate the prolactin content of pituitary preparations and of other tissues containing relatively high concentrations of this hormone (a minimum of about 0.03 IU or 2 μg of NIH-P-B1, 13 IU/mg); however, it was not possible to assay the prolactin present in serum from normal animals. Recently, double antibody radioimmunoassay techniques have been developed for ovine (2) and bovine prolactin (3) and a radioimmunoassay for rat prolactin (4) which uses a chromatoelectrophoresis step to separate free prolactin-125 I from that bound to the antibody has also been reported. These techniques are sufficiently sensitive to quantitate serum levels of prolactin. This communication describes the development of a double antibody radioimmunoassay for rat prolactin capable of measuring the amount of prolactin in 100 μl of serum from immature female rats or in 25 μl of serum from normal females during diestrus. Methods and Materials. Purified rat prolactin (HIV-8-C) for radioiodination and for immunization was prepared from whole, frozen rat pituitary glands by Ellis et al. (5). This preparation is designated B329 in the remainder of the present study. Other preparations with differing concentrations of rat prolactin were obtained by physiologic manipulation of three groups of mature, female Sprague-Dawley rats. Rats in the first group (B350) were ovariectomized and injected subcutaneously with 10 μg of estradiol benzoate in corn oil daily for 10 days. Rats in the second group (B349) were ovariectomized and injected with corn oil for 10 days while the third group was composed of untreated intact females during diestrus (B351). At the end of 2 weeks the rats were bled terminally via the abdominal vena cava, and the blood from each group was pooled and serum was obtained by centrifugation.