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.
Lewis
L K
LK
Cardioendocrine Research Group, Christchurch Hospital, New Zealand. llewis@chmeds.ac.nz
Smith
M W
MW
Yandle
T G
TG
Richards
A M
AM
Nicholls
M G
MG
D053607:Adrenomedullin; D000327:Adsorption; D000682:Amyloid; D000818:Animals; D000906:Antibodies; D001793:Blood Preservation; D015740:Calcitonin Gene-Related Peptide; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003429:Cross Reactions; D006461:Hemolysis; D006801:Humans; D058228:Islet Amyloid Polypeptide; D010446:Peptide Fragments; D010455:Peptides / Q000097:blood*; D011863:Radioimmunoassay / Q000379:methods; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine
D000682:Amyloid; D000906:Antibodies; D058228:Islet Amyloid Polypeptide; D010446:Peptide Fragments; D010455:Peptides; D053607:Adrenomedullin; D012710:Serum Albumin, Bovine; D015740:Calcitonin Gene-Related Peptide