Adrenomedullin(1–52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage

We describe a specific and sensitive RIA for human adrenomedullin (AM)(1-52). The detection limit and the concentration required for 50% inhibition of binding were 0.1 and 1.2 fmol/tube, respectively. Cross-reactivities with AM(1-12), AM(13-52), calcitonin gene-related peptide, amylin, and other vasoactive hormones were negligible. AM immunoreactivity in normal subjects ranged from 2.7 to 10.1 pmol/L (n = 44). We investigated factors influencing the recovery and measurement of AM in the assay. Recovery of labeled AM (> 80%) was markedly higher than that of unlabeled AM (56%). Immunoreactivity of exogenous AM added to plasma decreased up to 70% over four freeze-thaw cycles, whereas endogenous AM was stable. Alkali-treated casein (1 g/L) reduced adsorption of AM to surfaces and significantly increased assay precision compared with bovine serum albumin (P < 0.0001). HPLC separation of extracted plasma verified the presence of AM(1-52). We suggest that considerable care is needed to ensure that accurate and reproducible results are obtained from studies quantifying this peptide.