A double antibody radioimmunoassay was developed using a rabbit antiserum to rat prolactin (9.6 IU/mg) prepared from fresh frozen pituitary glands by (NH4)2SO4 extraction and precipitation followed by extraction and precipitation with ethanol. Further purification by gel filtration on Sephadex G-100 yielded prolactin for radioiodination and to serve as the reference standard (45.7 IU/mg). Standard curves were constructed over the range of 0.2–2.0 ng. Radioimmunoassay and pigeon crop sac potency estimates of crude and purified pituitary extracts were in excellent agreement. Rat FSH, LH, TSH and GH did not react in this assay. Plasma prolactin levels in hypophysectomized rats were >2.0 ng/ml. Plasma prolactin levels (ng/ml) in rats with 5-day estrous cycles were: 3 PM diestrus day 1, 3.3; 3 PM diestrus day 2, 4.8; 3 PM diestrus day 3, 9.1; 10 AM proestrus, 3.5; 3 PM proestrus, 44.6; 10 AM estrus, 9.3; 3 PM estrus, 7.5. The rat prolactin radioimmunoassay kit distributed by the National Institute of Arthritis and Metabolic Diseases (NIAMD) also has been examined. Although validating information is not provided by them, it exhibits excellent agreement between immunologic and biologic activities and is free from cross-reaction with FSH, LH, TSH and growth hormone. Prolactin levels in the serum of cycling rats measured by the NIAMD radioimmunoassay are virtually identical with those observed with our assay but higher concentrations of prolactin-like material were observed in serum of hypophysectomized rats in the NIAMD assay. (Endocrinology88: 548, 1971)
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Neill et al. (1971) studied this question.