Development of a radioimmunoassay for some agonists of growth hormone-releasing hormone.

Abstract

A radioimmunoassay (RIA) method was developed for determination of superactive GH-RH agonist Dat1,Ala15,Nle27 GH-RH(1-28)Agm29 (MZ-2-51) and some of the related analogs in biological fluids. The analogs were radioiodinated using the Bolton-Hunter method. For the generation of antibodies, rabbits were immunized with MZ-2-51 and its C-terminal derivative Nle27 GH-RH(17-28)Agm29, conjugated to bovine serum albumin with glutaraldehyde. The resulting antibodies exhibited high affinity and very low cross-reactivity with related, naturally occurring peptides, enabling us to set up a sensitive and specific RIA. High cross-reactions with some of the MZ-2-51 derivatives like MZ-3-149 (40%) and related compounds made it possible for us to also assay these analogs with the same antibody. At B/Bo of 23-37%, the final dilutions of the antibodies ranged from 1:35000 to 1:120000. The minimal detectable concentration of MZ-2-51 was 1.4 fmol (4.6 pg)/tube. The intra- and inter-assay variations were 2.2-4.1% and 9.3-13.9%, respectively. The antibody permitted direct determination of the analogs, without extraction, from biological fluids and tissue extracts. The analogs proved to be stable in serum, and no special treatment of sample was required. Pharmacodynamic studies were performed in rats. Serum levels of GH-RH(1-29) and two of its analogs were monitored following subcutaneous injection. Serum concentration of the analogs and GH-RH(1-29) reached a peak 15 min after injection and returned to basal levels within 90-120 min. Serum GH levels also reached a peak in 15 min, but declined more slowly in the case of analogs than GH-RH(1-29). The biological half-life of both analogs was significantly longer than that of GH-RH(1-29), probably due to their reduced enzymatic degradation.