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Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme
Arjun Trivedi1, Sandra Babic, Jean-Pierre Chanoine
1Department of Pediatrics, British Columbia Children's Hospital and Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada, V6H 3V4. atrivedi@interchange.ubc.ca
Objectives:
To investigate the effect of hemolysis and protease inhibitors on acylated ghrelin (AG) concentrations measured with a double antibody enzyme immunometric assay that uses an acetylcholinesterase (AChE)-Fab' conjugate.
Design And Methods:
Samples were hemolysed or treated with PHMB (p-hydroxymercuribenzoate), PMSF (phenylmethanesulfonylfluoride) or AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride) to prevent AG degradation.
Results:
Hemolysis decreased AG concentrations. PHMB or PMSF did not affect the assay. The standard curve was abolished by AEBSF but rescued by addition of a washing step prior to the AChE-Fab' conjugate.
Conclusions:
Hemolysis and AEBSF may affect AG determination.
