William E. Ellinwood

A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.

The measurement of neuropeptide Y in discrete hypothalamic and limbic regions of male rhesus macaques with a human NPY-directed antiserum

The distribution of neuropeptide Y (NPY) in microdissected brain regions of male macaques was quantified with a specific radioimmunoassay (RIA). The RIA consisted of a specific antiserum (R31) against human NPY that could detect 7 pg/tube with an IC50 of 125 pg/tube at a final dilution of 1:20,000. Varying amounts of rabbit and monkey mediobasal hypothalami yielded parallel [125I]NPY displacement curves in the assay and similar chromatographic elution profiles with those of synthetic human NPY. The NPY activity in acid extracts of discrete brain regions in castrate and castrated-testosterone-treated rhesus males was highest in mediobasal hypothalamus, followed by more rostral hypothalamic regions and amygdaloid nuclei. Testosterone did not alter NPY levels in any of the brain areas that we examined.