Development of specific antisera and a radioimmunoassay procedure for the gonadotropin-releasing hormone associated peptide (GAP) of the LHRH prohormone

Abstract

Recently, the structure of the cDNAs encoding the human and rat LHRH prohormones were determined and the corresponding peptide sequences deduced. In addition to LHRH, both cDNAs encoded a 56 amino acid sequence which has been designated gonadotropin-releasing hormone associated peptide (GAP). In order to initiate studies on the LHRH prohormone, three antisera (MC-1, MC-2 and MC-3) were generated using the human GAP (hGAP) 25–53 fragment as immunogen and a corresponding radioimmunoassay procedure was developed. The binding of 125I-labeled hGAP 1–56 to all three antisera was displaced completely by unlabeled hGAP 1–56. Acid extracts of rat median eminence produced displacement curves that were parallel to the hGAP 1–56 curve. Conversely, extracts of rat cortex and a number of brain and pituitary peptides, including LHRH and LHRH analogs, did not affect tracer binding. Several fragments of the human and rat GAP (rGAP) sequences (hGAP 25–53, hGAP 27–40 and rGAP 20–43) produced tracer displacement curves that were non-parallel with the hGAP 1–56 curve and/or gave only incomplete tracer displacement when all three antisera were tested. This suggests that the antisera contain multiple antibody populations directed toward several different antigenic determinants within the mid portion of the hGAP and rGAP sequences. The rGAP fragments, rGAP 1–11 and rGAP 39–53, failed to displace the tracer, further supporting that the antigenic sites occur within the mid portion of the rGAP sequence. Co-existence of radioimmunoassayable LHRH and GAP-like immunoreactivity (using MC-2 antiserum) was demonstrated in extracts of brain regions containing LHRH cell bodies, fibres and termini (preoptic area, hypothalamus, and median eminence). Radioimmunoassays employing these antisera, in conjunction with radioimmunoassays for LHRH, will be useful for studies of LHRH-prohormone localization, synthesis and processing under various physiologic and pathologic conditions.