Development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone

Abstract We describe the development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone (GHRH) based on the affinity purification of polyclonal rabbit antisera to rat GHRH using a human 1-29 GHRH affinity column. Assay sensitivity is 3.2 pg/ml using 100 mul of unextracted sample and the working range for the assay within 15% confidence limits is 64 to 5,000 pg/ml. Rat hypothalamic extract and secreted material demonstrated a single large peak of immunoreactive material coeluting with synthetic rat GHRH on high-performance liquid chromatography with a smaller, earlier peak which probably represents methionine sulphoxide [Met(O)(27)] GHRH. Extracted material diluted in parallel to the standard curve. Incubated rat hypothalami readily released measurable amounts of rat GHRH which responded appropriately to depolarization with 60 mM K(+) in a Ca(2+)-dependent manner.