The Effects of Rat Corticotrophin‐Releasing Factor‐41 Peptide Fragments on Bioassay and Immunoassay Determination of Corticotrophin‐Releasing Factor‐41 Levels

Abstract

Peptide fragments of rat corticotrophin-releasing factor-41 (CRF-41) containing amino-acid residues 21-33 antagonized the 5 nmolll CRF-41-stimulated adrenocorticotrophin secretion from the adult rat pituitary gland in vifro. The CRF 6-33 sequence had antagonistic effects at equimolar (5 nmolll) concentrations which were not observed at high (50 nmol/l) concentrations whilst the CRF 21-41 sequence had effects only at high (50 nmol/l) concentrations. Similar effects were observed with CRF 6-33 on basal release of adrenocorticotrophin. Peptide fragments elevated radioimmunoassay measurement of CRF-41 whilst inhibiting measurement of CRF-41 in a two-site enzyme amplified immunometric assay. The inhibitory effects of peptide fragments in the enzyme amplified immunometric assay could be removed by dilution to within the lower end of the standard curve or by increasing t h e concentration of antibody bound to the solid phase. These inhibitory effects mimic those of peptide fragments on basal adrenocorticotrophin release seen in a rat pituitary gland in vitro bioassay indicating that such two-site immunoassay determinations bear closer relation to bioactivity than those obtained using radioimmunoassay.Corticotrophin-releasing factor-41 (CRF-41) is a 41-residue peptide originally isolated from ovine hypothalami (1). The characterization of ovine CRF-41 revealed that the full length peptide is required for maximal stimulation of adrenocorticotrophin (ACTH) release from rat pituitary cells ( 2 ) . Rat CRF-41 differs from ovine CRF-41 in 7 of the 41 residues (3) but has similar bioactive properties. A range of substances including vasopressin and adrenaline exhibit corticotrophin-releasing activity which has complicated the interpretation of bioassay measurement, and CRF-41 levels in biologicdl samples are now predominantly d m a t e d using immunoassay techniques (4-9). However, immunoreactivity does not necessarily imply the presence of bioactivity and bioassay still has an essential role to play in physiological studies.Characterization of the CRF-41 receptor in the rat pituitary zland has shown high affinity receptors from which rat and ovine xquences are equipotent at displacing labelled ovine CRF-41 (10-12). Peptide fragments comprising rat 1-20 and ovine 21-41 do not displace labelled ovine CRF-41 and lack the ability to stimulate ACTH release (10, 13). Cross-reactivity studies for immunoassays have shown the ability of peptide fragments of CRF-41 to potentially elevate RIA measurement of CRF-41 levels. The sequence of CRF-41 contains a number of potential enzymatic cleavage sites which could lead to the generation of peptide fragments in biological samples (13). In this study we sought to compare the effects of synthetic peptide fragments of rat CRF-41 on bioassay, RIA and two-site enzyme amplified immunometric assay (EAIA) measurement of rat CRF-41. The bioassay chosen used freshly isolated fragments of anterior pituitary in a static incubation (14, 15). The two RIAs used polyclonal antisera which showed di...