Development and validation of a specific radioimmunoassay for PHI in plasma

A sensitive and specific radioimmunoassay for the recently isolated neuropeptide with N-terminal histidine and C-terminal isoleucine amide (PHI) has been developed which can detect 3.0 pmol/l of the peptide in plasma. The routine antiserum produced in rabbit had a titre of 1:800 000 and recognized the sequence 3-8 of the 27 amino acid peptide. Labelling of PHI with 125I was performed by the chloramine T method, and labelled PHI was separated from unlabelled on an octadecylsilyl (ODC)-silica column. Non-specific interference in the assay was excluded by extraction of plasma with ethanol to a mean recovery of 82.4%. Plasma samples diluted parallel to the standard curve and behaved as PHI on ODC-silica column. The intra-assay and inter-assay coefficient of variation (CV) values at a level of 24.0 pmol/l were 6.3% and 13.1%, respectively. In 75 normal adults, the fasting PHI concentration ranged from 3.5-30.0 pmol/l with a mean of 14.2 pmol/l. In 235 children, the PHI concentration varied with age. Ingestion of a meal caused a rapid and short-lived increase in the PHI concentration.