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Development and Validation of a Two‐Site Immunochemiluminometric Assay for Rat Growth Hormone‐Releasing Hormone
Author(s) -
Page M. D.,
Lewis M. D.,
Lewis B. M.,
Weeks I.,
Scanion M. F.
Publication year - 1989
Publication title -
journal of neuroendocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.062
H-Index - 116
eISSN - 1365-2826
pISSN - 0953-8194
DOI - 10.1111/j.1365-2826.1989.tb00143.x
Subject(s) - polyclonal antibodies , antiserum , assay sensitivity , medicine , endocrinology , hormone , radioimmunoassay , chemistry , growth hormone–releasing hormone , peptide hormone , chromatography , standard curve , growth hormone , biology , antibody , immunology , alternative medicine , pathology
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 μl 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.