Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

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Abstract
Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor [hpGRF(1-44)NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OH] were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund''s adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster injection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioimmunoassays for these peptides. By using antibodies with an apparent Ka of 3.3 .times. 10-12 (human) and 7.7 .times. 10-11 (rat), the sensitivity of these assays in both human and rat was found to be < 1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOH-terminal region of the molecule, as shown by its crossreactivity with various hpGRF analogs: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(1-40)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 125I-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 .+-. 0.2 for the human and 11.1 .+-. 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2-16 wk and a correlation between increasing body weight and GRF content. These radioimmunoassays will serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.